Compositions and Methods for Muscle Regeneration Using Prostaglandin E2

ABSTRACT

Provided herein are compositions, methods, and kits for proliferating muscle cells by exposing the muscle cells to a prostaglandin E2 (PGE2) compound or compound that activates PGE2 signaling. Also provided are methods for regenerating muscle in a subject suffering from muscular atrophy, dystrophy, and/or injury by administering a PGE2 compound alone or in combination with isolated muscle cells. The PGE2 compound in combination with the isolated muscle cells can be administered prophylactically to prevent a muscle disease or condition.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 15/891,278filed Feb. 7, 2018 (allowed); which is a continuation of U.S. Ser. No.15/498,293 filed Apr. 26, 2017, now U.S. Pat. No. 9,918,994 issued onMar. 20, 2018; which is a continuation of PCT/US2017/020650 filed Mar.3, 2017; which claims priority to U.S. Provisional Application Nos.62/303,979 filed Mar. 4, 2016 and 62/348,116 filed Jun. 9, 2016; thefull disclosures which are incorporated herein by reference in theirentirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No.AG020961, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Aug. 21, 2019, is named079445-1153470_SL.txt and is 2,985 bytes in size.

BACKGROUND OF THE INVENTION

In skeletal muscles, aging leads to progressively impaired regenerationand loss of muscle mass, strength and function. Loss of muscle functionis a major public health problem that often leads to severe loss ofmobility and impaired quality of life in the ever-increasing agedpopulation. A major determinant of muscle functional decline is theimpaired ability of skeletal muscle stem cells (MuSCs) to regeneratemuscle after acute injury or damage in the course of aging. There isalso a need to augment muscle regeneration in muscles that haveundergone damage, injury, and/or atrophy due to, for example,postoperative immobilization or disuse, cancer and HIV cachexia,muscular dystrophies, acute injury, and aging. Resident MuSCs are rarebut essential to the maintenance and repair of muscle, e.g., skeletalmuscle, smooth muscle and cardiac muscle throughout adulthood. Withaging, the number of functional stem cells declines and thus, the needto enhance the numbers and function of MuSCs increases.

Prostaglandin E2 (PGE2), also known as dinoprostone, has been employedin various clinical settings including to induce labor in women and toaugment hematopoietic stem cell transplantation. PGE2 can be used as ananticoagulant and antithrombotic agent. PGE2's role as a lipid mediatorthat can resolve inflammation is also well known. Nonsteroidalanti-inflammatory drugs (NSAIDs), inhibitors of COX-1 and/or COX-2,suppress inflammation by inhibiting prostanoids, mainly via PGE2biosynthesis.

PGE2 is synthesized from arachidonic acid by a cyclooxygenase (COX) andprostaglandin E synthase enzymes. Levels of PGE2 are physiologicallyregulated by the PGE2 degrading enzyme, 15-hydroxyprostaglandindehydrogenase (15-PGDH). 15-PGDH catalyzes the inactivating conversionof the PGE2 15-OH to a 15-keto group.

There remains a need in the art for effective treatments forregenerating or rejuvenating damaged, impaired, dysfunctional, and/oratrophied muscle in a subject in need thereof. The present inventionsatisfies this need and provides advantages as well.

BRIEF SUMMARY OF THE INVENTION

In one aspect of the present invention, provided herein is a method forstimulating the proliferation of a population of isolated muscle stemcells. The method includes culturing the population of isolated musclecells with a compound selected from the group consisting ofprostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, acompound that attenuates PGE2 catabolism, a compound that neutralizesPGE2 inhibition, a derivative thereof, an analog thereof, and acombination thereof.

In a second aspect of the present invention, provided herein is acomposition comprising a population of isolated muscle cells and acompound selected from the group consisting of prostaglandin E2 (PGE2),a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2catabolism, a compound that neutralizes PGE2 inhibition, a derivativethereof, an analog thereof, and a combination thereof.

In a third aspect of the present invention, provided herein is a kitcomprising the composition comprising a population of isolated musclecells and a compound selected from the group consisting of prostaglandinE2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound thatattenuates PGE2 catabolism, a compound that neutralizes PGE2 inhibition,a derivative thereof, an analog thereof, and a combination thereof, andan instruction manual.

In a fourth aspect, provided herein is a method for regenerating apopulation of muscle cells in a subject having a condition or diseaseassociated with muscle damage, injury, or atrophy. The method includesadministering to the subject a therapeutically effective amount of acompound selected from the group consisting of prostaglandin E2 (PGE2),a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2catabolism, a compound that neutralizes PGE2 inhibition, a derivativethereof, an analog thereof, and a combination thereof, and apharmaceutically acceptable carrier, to increase the population ofmuscle cells and/or to enhance muscle function in the subject.

In a fifth aspect, provided herein is a method for preventing ortreating a condition or disease associated with muscle damage, injury oratrophy in a subject in need thereof. The method includes administeringto the subject (i) a therapeutically effective amount of a compoundselected from the group consisting of prostaglandin E2 (PGE2), a PGE2prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2catabolism, a compound that neutralizes PGE2 inhibition, a derivativethereof, an analog thereof, and a combination thereof, and apharmaceutically acceptable carrier, and (ii) a population of isolatedmuscle cells, to prevent or treat the condition or disease associatedwith muscle damage, injury, or atrophy.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H show that transient PGE2 treatment promotes young MuSCproliferation in vitro. FIG. 1A: PGE2 levels after young tibialisanterior (TA) muscle injury (notexin, NTX); controls are uninjuredcontralateral TAs assayed by ELISA; (n=4 mice per timepoint). FIG. 1B:Expression of PGE2 synthesizing enzymes (Ptges2 and Ptges) by MuSCsafter notexin injury by RT-qPCR, (n=3 mice per timepoint). FIG. 1C:Increase in MuSC numbers after 24 hr treatment with vehicle (−) or PGE2(10 ng/ml), and subsequent culture on hydrogel until day 7 (acutetreatment); (n=12 mice in 4 independent experiments). FIG. 1D: Increasein MuSC numbers after transient 24 hr treatment with vehicle (−) or PGE2(10 ng/ml) in absence or presence of EP4 antagonist (ONO-AE3-208, 1 μM);(n=9 mice assayed in 3 independent experiments). FIGS. 1E-1G:Proliferation of EP4 null MuSCs. EP4^(f/f) (null) MuSCs were transducedwith a lentiviral vector encoding GFP/luciferase and treated withlentiviral vector encoding Cre (+Cre) or without (−Cre; empty vector) todelete EP4 alelles. Subsequently MuSCs were treated with vehicle (−) orPGE2 (10 ng/ml) for 24 hr and cultured on hydrogels for three days. FIG.1E: Scheme depicting EP4-null MuSC analysis. FIG. 1F: EP4 null MuSCnumbers; (n=6 mice in 2 independent experiments).

FIG. 1G: Representative image. Bar=40 μm GFP, green; mCherry, red. FIG.1H: MuSC numbers after culture in charcoal stripped medium treated withvehicle (−) or PGE2 (10 ng/ml) every two days for 7 days on hydrogels;(n=3 mice with 3 technical replicates). *P<0.05, **P<0.001, ***P<0.0005****P<0.0001. ANOVA test with Bonferroni correction for multiplecomparisons (FIGS. 1A, 1B, ID, and IF); paired t-test (FIG. 1C);Mann-Whitney test (FIG. 1H). Means+s.e.m. n.s., non-significant.

FIGS. 2A-2J show an aberrant response of aged MuSCs to PGE2. FIG. 2A:PGE2 levels after aged TA injury (notexin, NTX); controls are uninjuredcontralateral TAs assayed by ELISA; (n=4 mice per timepoint). FIG. 2B:PGE2 levels in TAs of uninjured young (n=7 mice) and aged (n=5 mice)mice assayed by ELISA. FIG. 2C: Scheme showing PGE2 catabolism viadegrading enzyme 15-PGDH to its inactive PGE metabolite,13,14-dihydro-15-keto PGE2 (PGEM). FIG. 2D: Levels of PGEM quantified bymass spectrometry; (n=4 mice per age group). FIG. 2E: Expression of PGE2degrading enzyme 15-PGDH (Hpgd); (n=3 mice with 2 technical replicates).FIG. 2F: Increase in aged MuSC numbers after acute 24 hr treatment withvehicle (−), PGE2 (10 ng/ml) or the 15-PGDH inhibitor, SW033291 (1 μM;SW) assayed at day 7; (n=15 mice in 5 independent experiments). FIG. 2G:Aged MuSC numbers after culture in charcoal stripped medium treated withvehicle (−) or PGE2 (10 ng/ml) every two days for 7 days on hydrogels;(n=3 mice with 3 technical replicates). FIG. 2H: Scheme depicting PGE2effects on MuSCs. PGE2 acts through the EP4 receptor/cAMP (cyclic AMP)signaling pathway to promote proliferation. In aged MuSCs, followingintracellular transport by PGT (Prostaglandin transporter), PGE2catabolism is mediated by 15-PGDH to the inactive form, PGEM. FIG. 2I:Trajectories from a clone of aged MuSCs tracked by time-lapse microscopyfor 48 h in a microwell for control (left) and after acute treatmentwith PGE2 (right). The trajectory of the original cell and each of itsnewborn progeny are represented by a different color. FIG. 2J: Change inaged MuSC live cell counts (numbers) in clones tracked by time-lapsemicroscopy for control (left, n=32 clones) and after acute treatmentwith PGE2 (right, n=45 clones). The proportion of live cells in eachgeneration (G1-G6) at all timepoints is shown as cell number normalizedto a starting population of 100 single MuSCs. The percent increase inlive cell count was 4.0% (control) and 5.4% (PGE2-treated) (top panels).Change in aged MuSC dead cell counts (numbers) in clones tracked bytime-lapse microscopy for control (left) and after acute treatment withPGE2 (right). The proportion of dead cells in each generation (G1-G6) atall timepoints is shown as cell number normalized to a startingpopulation of 100 single MuSCs. The percent increase in dead cell countwas 1.0% (control) and 0.1% (PGE2-treated) (bottom panels). *P<0.05,**P<0.001, ***P<0.0005. ANOVA test with Bonferroni correction formultiple comparisons (FIGS. 2A and 2F); Mann-Whitney test (FIGS. 2B, 2D,2E, and 2G). Means±s.e.m. n.s., non-significant.

FIGS. 3A-3D show that acute PGE2 treatment promotes MuSC engraftment andregeneration in vivo. FIG. 3A: Engraftment of cultured GFP/luc-labeledyoung MuSCs (250 cells) isolated from transgenic mice after acutetreatment with vehicle (−) or PGE2 as described in FIG. 1C. Transplantscheme (top). Non-invasive bioluminescence imaging (BLI) signal measuredas radiance for each TA; (n=5 mice per condition) (bottom). FIG. 3B:Engraftment of GFP/luc-labeled EP4^(f/f) MuSCs (1,000 cells) treatedwith Cre (+Cre) or without (−Cre; empty vector) in culture to delete EP4alelles. EP4i MuSCs were transduced with a lentiviral vector encodingGFP/luciferase for BLI. Transplant scheme (top). BLI signalspost-transplant (n=5 mice per condition (bottom). FIG. 3C: Engraftmentof freshly sorted GFP/luc-labeled young MuSCs (250 cells) coinjectedwith vehicle (−) or dmPGE2. Transplant scheme (top). BLI signalspost-transplant; (n=4 and n=5 mice for vehicle and dmPGE2 treated,respectively). FIG. 3D: Engraftment of GFP/luc-labeled aged MuSCs (250cells) coinjected with vehicle (−) or dmPGE2; (n=3 mice per condition)(bottom). Aged MuSCs were transduced with a lentiviral vector encodingGFP/luciferase for BLI. Transplant scheme (top). BLI signalspost-transplant expressed as average radiance (p s⁻¹ cm⁻² sr⁻¹).Representative BLI images for each condition. Bar=5 mm (FIGS. 3A-3D).Data are representative of two independent experiments. * P<0.05,**P<0.001 and ***P<0.0005. ANOVA test for group comparisons andsignificant difference for endpoints by Fisher's test. Means+s.e.m.

FIGS. 4A-4P show that intramuscular injection of PGE2 alone promotesMuSC expansion, improves regeneration, and increases force. Young:(FIGS. 4A-4D) TA muscles of young mice were injected with vehicle (−) ordmPGE2 48 hr post-cardiotoxin (CTX) injury; (n=3 mice per condition).FIG. 4A: Scheme of experimental procedure (top). Representative TAcross-section (bottom) with nuclei (DAPI; blue), LAMININ (green) andPAX7 (red) staining 14 days after cardiotoxin injury. Arrowheadsindicate PAX7⁺ MuSCs. Bar-40 μm. FIG. 4B: Increase in endogenous MuSCsby immunofluorescence of PAX7 expressing satellite cells per 100 fibersin cross-sections of TAs from young mice. FIG. 4C: Myofibercross-sectional areas (CSA) in vehicle (−, open white bar) and dmPGE2treated (filled blue bar) young TAs quantified using the BaxterAlgorithms for Myofiber Analysis. FIG. 4D: Distribution of small (<1,000μm² CSA) and large (>1,000 μm² CSA) myofibers. (FIGS. 4E-4G) Increase inendogenous MuSCs assayed by Pax7-luciferase. Pax7^(CreERT2);Rosa26-LSL-Luc mice were treated intraperitoneally with tamoxifen (TAM),TAs subjected to cardiotoxin (CTX) injury, injected with vehicle (−) ordmPGE2 3 days later and monitored by BLI; (n=3 mice per condition). FIG.4E: Scheme of experimental procedure. FIG. 4F: BLI (n=3 mice percondition). FIG. 4G: Representative BLI image. Bar=5 mm. Aged: (FIGS.4H-4K) TAs of aged mice were treated in vivo with vehicle (−) or dmPGE2treatment 48 hr post-cardiotoxin (CTX) injury; (n=3 mice per condition).FIG. 4H: Scheme of experimental procedure (top). Representative TAcross-section (bottom) with nuclei (DAPI;blue), LAMININ (green) and PAX7(red) staining 14 days after cardiotoxin injury. Arrowheads indicatePAX7⁺ muscle stem cells. Bar=40 μm. FIG. 4I: Increase in endogenousMuSCs as in FIG. 4B for aged mice. FIG. 4J: Myofiber cross-sectionalarea (CSA) as in FIG. 4C for aged TAs. FIG. 4K: Distribution of CSA asin FIG. 4D for aged TAs. (FIGS. 4L-4P) Increase in strength in aged micemeasured in vivo as muscle contractile force after downhill treadmillrun. Mice were subject to a 20° downhill treadmill run for 2 consecutiveweeks and force was assayed at week 5. During the first week, medial andlateral gastrocnemius (GA) of aged mice were injected either withvehicle (−) or dmPGE2. n=10 or 8 biological replicates for vehicle (−)treated or dmPGE2 treated, respectively, with 5 technical replicateseach. FIG. 4L: Experimental scheme. Representative twitch force (FIG.4M) and tetanic force (FIG. 4N). Specific muscle twitch forces (FIG. 4O)and specific muscle tetanic force (FIG. 4P) were calculated bynormalizing force to physiological cross sectional areas (PCSA). Pairedt-test (FIGS. 4B, 4D, 4I and 4K); ANOVA test for group comparison andsignificant difference for the endpoint by Fisher's test (FIG. 4F);Mann-Whitney test (FIGS. 40 and 4P). *P<0.05, **P<0.001 and****P<0.0001. Means+s.e.m.

FIGS. 5A-5K show that PGE2 promotes MuSC expansion. FIG. 5A: PGE2 levelsday 3 after cryoinjury for tibialis anterior (TA) hindlimb muscles ofyoung mice compared to contralateral uninjured controls as assayed byELISA; (n=4 mice per time point per condition). FIG. 5B: Representativeimage of dividing muscle stem cells (MuSCs) labelled with EdU (red)during 1 hr after treatment with PGE2 (10 ng/ml) for 24 h (d0 to d1) orvehicle (−), and stained for MYOGENIN (green). Bar represents 40 m. FIG.5C: Percentage of dividing MuSCs labeled with EDU as in (b); (n=6 micewith 3 technical replicates in two independent experiments). FIG. 5D:Increase in proliferation measured by the metabolic viability assayVisionBlue after treatment with vehicle (−) or indicated doses of PGE2(1-200 ng/ml); (n=6 mice with 3 technical replicates in two independentexperiments). FIG. 5E: Expression of prostaglandin receptors (Ptger 1-4)by MuSCs after 24 hr treatment with vehicle (−) or PGE2; (n=3 mice with2 technical replicates). FIG. 5F: Increase in cAMP levels in MuSCs after1 hr PGE2 treatment relative to untreated controls (−); (n=6 mice with 3technical replicates assayed in 2 independent experiments). FIGS. 5G-5H:Expression of Pax7 (FIG. 5G) and Myogenin (FIG. 5H) by MuSCs after 24 hrtreatment with vehicle (−) or PGE2; (n=3 mice with 2 technicalreplicates). FIGS. SI-5J: EP4^(f/f) MuSCs were transduced with alentiviral vector encoding GFP/luciferase and treated with lentiviralvector encoding Cre (+Cre) or without (−Cre; empty vector) to delete EP4alelles. Bar graphs show percentage of +Cre MuSCs (FIG. 5I) and GFP/LucMuSCs (FIG. 5J). FIG. 5K: Representative image of MuSCs in hydrogelculture after 7 days in myoblast medium containing charcoal strippedfetal bovine supplemented with vehicle (−) or PGE2 (10 ng/ml) every twodays. Bar represents 40 μm. *P<0.05, **P<0.001, ***P<0.0005. Pairedt-test (FIGS. SA, SE, 5G, and 5H); Mann-Whitney test (FIG. 5C).Means+s.e.m. n.s., non-significant.

FIGS. 6A-6C show mass spectrometry analysis of young and aged muscle todetect prostaglandins and PGE2 metabolites. FIG. 6A: Chemicalstructures, chemical formula, exact mass and molecular weight ofanalyzed prostaglandins (PGE2, PGF2α and PGD2) and PGE2 metabolites(15-keto PGE2 and 13,14-dihydro-15-keto PGE2). The internal standardsPGF2α-D9 and PGE2-D9 were added to all composite standards. FIG. 6B:Calibration lines for liquid chromatography-electrosprayionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis wereprepared by diluting stock solutions to final concentrations of 0.1ng/ml to 500 ng/ml. Standard curve equations and correlationcoefficients are shown for each standard. FIG. 6C: Representativechromatogram. The separate peaks show excellent chromatographicresolution of the analyzed prostaglandins and their metabolites. cps,counts per second.

FIGS. 7A-7G show that aged MuSCs increase proliferation and cellsurvival in response to PGE2 treatment. FIGS. 7A-7C: mRNA levelsmeasured by qRT-PCR were normalized to Gapdh for young and aged MuSCs;(n=3 mice with 2 technical replicates). FIG. 7A: Prostaglandintransporter (PGT) encoded by the Slco2a1 gene. FIG. 7B: PGE2synthesizing enzymes, Ptges and Ptges2. FIG. 7C: EP1-4 receptors encodedby the genes Ptger1-4. FIG. 7D: Pax7 mRNA levels in MuSCs after 24 hrtreatment with vehicle (−) or PGE2 treatment; (n=3 mice with 2 technicalreplicates). FIG. 7E: Single aged MuSC clones tracked by time-lapsemicroscopy after acute treatment with vehicle (−; top) or PGE2 (bottom).For each clone the resulting number of live (open bar) and dead (blackbar) cells after 48 h timelapse tracking is shown. FIG. 7F:Proliferation curve of tracked live aged MuSCs assessed by time-lapsemicroscopy for vehicle (−) or transient PGE2 treatment during 48 h. FIG.7G: Flow cytometry analysis of apoptotic Annexin V⁺ on aged MuSCs after24 hr treatment with vehicle (−) or PGE2 and analyzed 7 days later aftergrowth on hydrogels; (n=9 mice in 3 independent experiments).Mann-Whitney test (FIGS. 7A-7D) and paired t-test (FIG. 7G) at α=0.05.Means+s.e.m. n.s., non-significant.

FIGS. 8A-8B show Baxter Algorithms for Myofiber Analysis of musclecross-sectional area. FIG. 8A: Representative cross-sectional images oftibialis anterior myofibers of young mice treated in vivo with vehicle(−) or PGE2 48 hr post-cardiotoxin (CTX) injury. Images show stainingwith LAMININ, green and DAPI, blue. FIG. 8B: The correspondingsegmentation images from FIG. 8A analyzed by the Baxter Algorithms forMyofiber Analysis to determine the cross sectional area (CSA) oftransverse sections of myofibers (bottom) at day 14 post-injury. Barrepresents 40 μm.

FIGS. 9A-9G show that deletion of PGE2 receptor EP4 in MuSCs decreasesregeneration and force of skeletal muscle after injury. Tibialisanteriors (TAs) of Pax7-specific EP4 conditional knockout mice(Pax7^(CreERT2);EP4^(fl/fl)) treated with tamoxifen were assayed at 6(FIGS. 9C-9D), 21 (FIGS. 9B and 9E), and 14 (FIGS. 9F and 9G) dayspost-notexin injury; (n=3 mice per condition). FIG. 9A: Experimentalscheme. FIG. 9B: Expression of Ptger4 (EP4 receptor) in sorted MuSCs(α⁷⁺ CD34⁺ lin⁻) from control or EP4 KO mice post-injury. FIG. 9C:Representative TA cross-section. DAPI, blue; Embryonic Myosin HeavyChain (eMyHC), red. Bar=40 μm. FIG. 9D: Percentage of eMyHC⁺ fibers.FIG. 9E: Myofiber cross-sectional areas (CSA) in control andPax7-specific EP4 knockout TAs. FIG. 9F: Muscle twitch forces and (FIG.9G) muscle tetanic force at day 14 post-notexin injury. Mann-Whitneytest (FIGS. 9B, 9C, 9F, and 9G); ANOVA test for group comparison andsignificant difference for each bin by Fisher's test (FIG. 9E). *P<0.05, ***P<0.0005, and ****P<0.0001. Means+s.e.m.

FIGS. 10A-10C show that blockage of endogenous PGE2 signaling in muscleat an early timepoint of regeneration reduces regeneration and force.Endogenous MuSCs assayed in Pax7^(CreERT2); Rosa26-LSL-Luc mice treatedwith tamoxifen (TAM) by non-invasive bioluminescence imaging (BLI) afterinjection with vehicle (−) or NSAID (Indomethacin) post-cardiotoxininjury into the Tibialis anterior (TA); (n=3 mice per condition). FIG.10A: Experimental scheme. FIG. 10B: BLI; (n=3 mice per condition). FIG.10C: Muscle twitch forces at day 14 post-notexin injury (n=8 forvehicle-treated and 10 for NSAID-treated). ANOVA test for groupcomparison and significant difference for the endpoint by Fisher's test(FIG. 10B). Mann-Whitney test (FIG. 10C). * P<0.05, **P<0.001,***P<0.0005, and ****P<0.0001. Means+s.e.m

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based, in part, on the discovery thatprostaglandin E2 (PGE2) can improve muscle cell proliferation andfunction. PGE2 alone or in combination with isolated muscle cells can beused to repair muscle damage due to injury, atrophy, or disease. Infact, PGE2-treated muscle cells exhibit enhanced muscle regeneration andimproved muscle function upon administration. As such, provided hereinare novel therapeutic methods, compositions, and kits to promote muscleregeneration and rejuvenation of damaged, injured, or atrophied muscle.

II. General

Practicing this invention utilizes routine techniques in the field ofmolecular biology. Basic texts disclosing the general methods of use inthis invention include Sambrook and Russell, Molecular Cloning. ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb), base pairs(bp), or nucleotides (nt). Sizes of single-stranded DNA and/or RNA canbe given in nucleotides. These are estimates derived from agarose oracrylamide gel electrophoresis, from sequenced nucleic acids, or frompublished DNA sequences. For proteins, sizes are given in kilodaltons(kDa) or amino acid residue numbers. Protein sizes are estimated fromgel electrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized, e.g., according to the solid phase phosphoramidite triestermethod first described by Beaucage and Caruthers, Tetrahedron Lett.22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purificationof oligonucleotides is performed using any art-recognized strategy,e.g., native acrylamide gel electrophoresis or anion-exchange highperformance liquid chromatography (HPLC) as described in Pearson andReanier, J. Chrom. 255: 137-149 (1983).

III. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents knownto those skilled in the art, and so forth.

The term “prostaglandin E2” or “PGE2” refers to prostaglandin that canbe synthesized via arachidonic acid via cyclooxygenase (COX) enzymes andterminal prostaglandin E synthases (PGES). PGE2 plays a role in a numberof biological functions including vasodilation, inflammation, andmodulation of sleep/wake cycles.

The term “prostaglandin E2 receptor agonist” or “PGE2 receptor agonist”refers to a chemical compound, small molecule, polypeptide, biologicalproduct, etc. that can bind to and activate any PGE2 receptor, therebystimulating the PGE2 signaling pathway.

The term “compound that attenuates PGE2 catabolism” refers to a chemicalcompound, small molecule, polypeptide, biological product, etc. that canreduce or decrease the breakdown of PGE2.

The term “compound that neutralizes PGE2 inhibition” refers to achemical compound, small molecule, polypeptide, biological product, etc.that can block or impede an inhibitor of PGE2 synthesis, activity,secretion, function, and the like.

The term “derivative,” in the context of a compound, includes but is notlimited to, amide, ether, ester, amino, carboxyl, acetyl, and/or alcoholderivatives of a given compound.

The term “embryonic stem cell-derived muscle cell” or “ESC-derivedmuscle cell” refers to a muscle cell that is derived from ordifferentiated from an embryonic stem cell.

The term “induced pluripotent stem cell-derived muscle cell” or“iPSC-derived muscle cell” refers to a muscle cell that is derived fromor differentiated from an induced pluripotent stem cell.

The term “isolated,” in the context of cells, refers to a single cell ofinterest or a population of cells of interest, at least partiallyisolated and/or purified from other cell types or other cellularmaterial with which it naturally occurs in the tissue of origin (e.g.,muscle tissue). A population of muscle cells is “isolated” when it is atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98% and, incertain cases, at least about 99% free of cells that are not musclecells. Purity can be measured by any appropriate method, for example, byfluorescence-activated cell sorting.

The term “autologous” refers to any material (e.g., a cell) derived fromthe same individual to whom it is later to be re-introduced into theindividual.

The term “allogeneic” refers to any material (e.g., a cell) derived froma different animal of the same species as the individual to whom thematerial is introduced. Two or more individuals are said to beallogeneic to one another when the genes at one or more loci are notidentical. In some aspects, allogeneic material from individuals of thesame species may be sufficiently unlike genetically to interactantigenically.

The term “treating” or “treatment” refers to any one of the following:ameliorating one or more symptoms of disease; preventing themanifestation of such symptoms before they occur; slowing down orcompletely preventing the progression of the disease (as may be evidentby longer periods between reoccurrence episodes, slowing down orprevention of the deterioration of symptoms, etc.); enhancing the onsetof a remission period; slowing down the irreversible damage caused inthe progressive-chronic stage of the disease (both in the primary andsecondary stages); delaying the onset of said progressive stage; or anycombination thereof.

The term “administer,” “administering,” or “administration” refers tothe methods that may be used to enable delivery of agents orcompositions such as the compounds and cells described herein to adesired site of biological action. These methods include, but are notlimited to, parenteral administration (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular, intra-arterial, intravascular,intracardiac, intrathecal, intranasal, intradermal, intravitreal, andthe like), transmucosal injection, oral administration, administrationas a suppository, and topical administration. One skilled in the artwill know of additional methods for administering a therapeuticallyeffective amount of the compounds and/or cells described herein forpreventing or relieving one or more symptoms associated with a diseaseor condition.

The term “therapeutically effective amount” or “therapeuticallyeffective dose” or “effective amount” refers to an amount of a compound,therapeutic agent (e.g., cells), and/or pharmaceutical drug that issufficient to bring about a beneficial or desired clinical effect. Atherapeutically effective amount or dose may be based on factorsindividual to each patient, including, but not limited to, the patient'sage, size, type or extent of disease, stage of the disease, route ofadministration of the regenerative cells, the type or extent ofsupplemental therapy used, ongoing disease process and type of treatmentdesired (e.g., aggressive vs. conventional treatment). Therapeuticallyeffective amounts of a pharmaceutical compound or compositions, asdescribed herein, can be estimated initially from cell culture andanimal models. For example, IC₅₀ values determined in cell culturemethods can serve as a starting point in animal models, while IC₅₀values determined in animal models can be used to find a therapeuticallyeffective dose in humans.

The term “pharmaceutically acceptable carrier” refers to refers to acarrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, rats, simians, humans, farm animals, sport animals, and pets.

The term “acute exposure,” in the context of administration of acompound, refers to a temporary or brief application of a compound to asubject, e.g., human subject, or cells. In some embodiments, an acuteexposure includes a single administration of a compound over the courseof treatment or over an extended period of time.

The term “intermittent exposure,” in the context of administration of acompound, refers to a repeated application of a compound to a subject,e.g., human subject, or cells, wherein a desired period of time lapsesbetween applications.

The term “acute regimen,” in the context of administration of acompound, refers to a temporary or brief application of a compound to asubject, e.g., human subject, or to a repeated application of a compoundto a subject, e.g., human subject, wherein a desired period of time(e.g., 1 day) lapses between applications. In some embodiments, an acuteregimen includes an acute exposure (e.g., a single dose) of a compoundto a subject over the course of treatment or over an extended period oftime. In other embodiments, an acute regimen includes intermittentexposure (e.g., repeated doses) of a compound to a subject in which adesired period of time lapses between each exposure.

The term “continuous exposure,” in the context of administration of acompound, refers to a repeated, chronic application of a compound to asubject, e.g., human subject, or cells, over an extended period of time.

The term “chronic regimen,” in the context of administration of acompound, refers to a repeated, chronic application of a compound to asubject, e.g., human subject, over an extended period of time such thatthe amount or level of the compound is substantially constant over aselected time period. In some embodiments, a chronic regimen includes acontinuous exposure of a compound to a subject over an extended periodof time.

IV. Detailed Description of the Embodiments

In one aspect, provided herein is a method for stimulating theproliferation, expansion, and/or engraftment of a population of isolatedmuscle cells by culturing the population of isolated muscle cells with acompound selected from the group consisting of prostaglandin E2 (PGE2),a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2catabolism, a compound that neutralizes PGE2 inhibition, a derivativethereof, an analog thereof, and a combination thereof. In someembodiments, the population of isolated muscle cells is substantiallypurified or purified (e.g., separated from non-muscle cells or othercells that are not of interest). In some instances, the population ofisolated muscle cells comprises skeletal muscle cells, smooth musclecells, cardiac muscle cells, embryonic stem cell-derived muscle cells,induced pluripotent stem cell-derived muscle cells, dedifferentiatedmuscle cells, or a combination thereof. In particular embodiments, thepopulation of isolated muscle cells comprises muscle stem cells,satellite cells, myocytes, myoblasts, myotubes, myofibers, or acombination thereof.

The population of isolated muscle cells can be obtained from a subject.In other embodiments, the isolated muscles cells are from a cell line,e.g., a primary cell line. In some instances, the subject has acondition or disease associated with muscle damage, injury, or atrophy.The condition or disease associated with muscle damage, injury, oratrophy can be selected from the group consisting of acute muscleinjury, tear, or trauma, soft tissue hand injury, Duchenne musculardystrophy (DMD), Becker muscular dystrophy, limb girdle musculardystrophy, amyotrophic lateral sclerosis (ALS), distal musculardystrophy (DD), inherited myopathies, myotonic muscular dystrophy (MDD),mitochondrial myopathies, myotubular myopathy (MM), myasthenia gravis(MG), congestive heart failure, periodic paralysis, polymyositis,rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiaccachexia, stress induced urinary incontinence, and sarcopenia.

In some embodiments, the PGE2 derivative comprises 16,16-dimethylprostaglandin E2. In other embodiments, the compound that attenuatesPGE2 catabolism comprises a compound, neutralizing peptide, orneutralizing antibody that inactivates or blocks 15-hydroxyprostaglandindehydrogenase (15-PGDH) or inactivates or blocks a prostaglandintransporter (PTG or SLCO2A1), which transports PGE2 inside the cells forcatabolism by 15-PGDH.

In some embodiments, the step of culturing the population of isolatedmuscle cells with the compound comprises acute, intermittent, orcontinuous exposure of the population of isolated muscle cells to thecompound. The compound may be exposed to the isolated cells once in anacute manner. In other cases, the compound may be exposed to theisolated cells at more than one time point such that time elapsesbetween exposures. In yet other cases, the compound may be exposed tothe isolated cells continuously such that the level of compound indirect contact with the cells does not fall below a pre-selected amount.

In particular embodiments, provided herein is a method for promotingmuscle cell engraftment in a subject. The method includes culturing orcontacting a population of isolated muscle cells with an effectiveamount of a compound selected from the group consisting of prostaglandinE2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound thatattenuates PGE2 catabolism, a compound that neutralizes PGE2 inhibition,a derivative thereof, an analog thereof, and a combination thereof, topromote engraftment of the muscle cells in the subject; andadministering the cultured or contacted muscle cells to the subject.

In another aspect, provided herein is a composition comprising apopulation of isolated muscle cells and a compound selected from thegroup consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2receptor agonist, a compound that attenuates PGE2 catabolism, a compoundthat neutralizes PGE2 inhibition, a derivative thereof, an analogthereof, and a combination thereof. In some embodiments, the populationof isolated muscle cells comprises skeletal muscle cells, smooth musclecells, cardiac muscle cells, embryonic stem cell-derived muscle cells,induced pluripotent stem cell-derived muscle cells, dedifferentiatedmuscle cells, or a combination thereof. In some instances, thepopulation of isolated muscle cells comprises muscle stem cells,satellite cells, myocytes, myoblasts, myotubes, myofibers, or acombination thereof. The composition can also include a pharmaceuticallyacceptable carrier.

In yet another aspect, provided herein is a kit comprising any of thecompositions disclosed herein, and an instruction manual.

In another aspect, provided herein is a method for regenerating apopulation of muscle cells in a subject having a condition or diseaseassociated with muscle damage, injury, or atrophy. The method includesadministering to the subject a therapeutically effective amount of acompound selected from the group consisting of prostaglandin E2 (PGE2),a PGE2 prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2catabolism, a compound that neutralizes PGE2 inhibition, a derivativethereof, an analog thereof, and a combination thereof, and apharmaceutically acceptable carrier, to increase the population ofmuscle cells in the subject and/or to enhance muscle function in thesubject.

In a related aspect, provided herein is a method for stimulating theproliferation and/or expansion of a population of muscle cells in asubject having a condition or disease associated with muscle damage,injury, or atrophy. The method includes administering to the subject atherapeutically effective amount of a compound selected from the groupconsisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptoragonist, a compound that attenuates PGE2 catabolism, a compound thatneutralizes PGE2 inhibition, a derivative thereof, an analog thereof,and a combination thereof, and a pharmaceutically acceptable carrier, toincrease the population of muscle cells in the subject and/or to enhancemuscle function in the subject.

In some embodiments, the population of muscle cells comprises anendogenous population of muscle cells. In other embodiments, thepopulation of muscle cells comprises a population of isolated musclecells that has been administered (e.g., injected or transplanted) to thesubject. In yet other embodiments, the population of muscle cellscomprises both an endogenous population of muscle cells and a populationof isolated muscle cells that has been administered to the subject.

In some embodiments, the condition or disease associated with muscledamage, injury, or atrophy is selected from the group consisting ofacute muscle injury or trauma, soft tissue hand injury, Duchennemuscular dystrophy (DMD), Becker muscular dystrophy, limb girdlemuscular dystrophy, amyotrophic lateral sclerosis (ALS), distal musculardystrophy (DD), inherited myopathies, myotonic muscular dystrophy (MDD),mitochondrial myopathies, myotubular myopathy (MM), myasthenia gravis(MG), congestive heart failure, periodic paralysis, polymyositis,rhabdomyolysis, dermatomyositis, cancer cachexia, AIDS cachexia, cardiaccachexia, stress induced urinary incontinence, and sarcopenia.

In some embodiments, the population of muscle cells comprises skeletalmuscle cells, smooth muscle cells, cardiac muscle cells, embryonic stemcell-derived muscle cells, induced pluripotent stem cell-derived musclecells, dedifferentiated muscle cells, or a combination thereof. In somecases, the population of muscle cells comprises muscle stem cells,satellite cells, myocytes, myoblasts, myotubes, myofibers, or acombination thereof.

In some embodiments, the PGE2 derivative comprises 16,16-dimethylprostaglandin E2.

In some embodiments, the compound that attenuates PGE2 catabolismcomprises a compound, neutralizing peptide, or neutralizing antibodythat inactivates or blocks 15-hydroxyprostaglandin dehydrogenase(15-PGDH) or inactivates or blocks a prostaglandin transporter (PTG orSLCO2A1).

In some embodiments, the step of administering the compound comprisesoral, intraperitoneal, intramuscular, intra-arterial, intradermal,subcutaneous, intravenous, or intracardiac administration. In somecases, the compound is administered in accordance with an acute regimen.In certain instances, the acute regimen comprises acute exposure (e.g.,a single dose) of the compound to the subject. In other instances, theacute regimen comprises intermittent exposure (e.g., repeated doses) ofthe compound to the subject. As a non-limiting example, an acute PGE2regimen can comprise a series of intermittent (e.g., daily) doses ofPGE2 over a desired period of time (e.g., over the course of 2, 3, 4, 5,6, or 7 days).

In other embodiments, the step of administering further comprisesadministering a population of isolated muscle cells to the subject. Thepopulation of isolated muscle cells can be autologous to the subject.The population of isolated muscle cells can be allogeneic to thesubject. In some instances, the population of isolated muscle cells issubstantially purified or purified. In other instances, the populationof isolated muscle cells is cultured with the compound prior to beingadministered to the subject. The step of culturing the population ofisolated muscle cells with the compound can include acute, intermittent,or continuous exposure of the population of isolated muscle cells to thecompound. Administering the population of isolated muscle cells cancomprise injecting or transplanting the cells into the subject. Thepopulation of isolated muscle cells and the compound can be administeredto the subject concomitantly. Alternatively, the population of isolatedmuscle cells and the compound can be administered to the subjectsequentially.

In another aspect, provided herein is a method for preventing ortreating a condition or disease associated with muscle damage, injury oratrophy in a subject in need thereof. The method includes administeringto the subject (i) a therapeutically effective amount of a compoundselected from the group consisting of prostaglandin E2 (PGE2), a PGE2prodrug, a PGE2 receptor agonist, a compound that attenuates PGE2catabolism, a compound that neutralizes PGE2 inhibition, a derivativethereof, an analog thereof, and a combination thereof, and apharmaceutically acceptable carrier, and (ii) a population of isolatedmuscle cells, to prevent or treat the condition or disease associatedwith muscle damage, injury, or atrophy.

In a related aspect, provided herein is a method for stimulating theproliferation and/or expansion of a population of muscle cells in asubject having a condition or disease associated with muscle damage,injury, or atrophy by administering to the subject (i) a therapeuticallyeffective amount of a compound selected from the group consisting ofprostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, acompound that attenuates PGE2 catabolism, a compound that neutralizesPGE2 inhibition, a derivative thereof, an analog thereof, and acombination thereof, and a pharmaceutically acceptable carrier, and (ii)a population of isolated muscle cells. In some embodiments, thepopulation of muscle cells comprises an endogenous population of musclecells. In other embodiments, the population of muscle cells comprisesthe population of isolated muscle cells that has been administered(e.g., injected or transplanted) to the subject. In yet otherembodiments, the population of muscle cells comprises both an endogenouspopulation of muscle cells and the population of isolated muscle cellsthat has been administered to the subject. In certain embodiments, thetherapeutically effective amount of the compound comprises an amountthat is sufficient to increase the population of endogenous muscle cellsin the subject and/or to increase the population of isolated musclecells that has been administered to the subject and/or to enhance musclefunction in the subject.

In some embodiments, the PGE2 derivative comprises 16,16-dimethylprostaglandin E2. In some instances, the compound that attenuates PGE2catabolism comprises a compound, neutralizing peptide, or neutralizingantibody that inactivates or blocks 15-hydroxyprostaglandindehydrogenase (15-PGDH) or inactivates or blocks a prostaglandintransporter (PTG or SLCO2A1).

In some embodiments, the population of muscle cells comprises skeletalmuscle cells, smooth muscle cells, cardiac muscle cells, embryonic stemcell-derived muscle cells, induced pluripotent stem cell-derived musclecells, dedifferentiated muscle cells, or a combination thereof. In somecases, the population of muscle cells comprises muscle stem cells,satellite cells, myocytes, myoblasts, myotubes, myofibers, or acombination thereof. The population of isolated muscle cells can besubstantially purified or purified.

In some embodiments, the population of isolated muscle cells is culturedwith the compound prior to being administered to the subject. In somecases, culturing the population of isolated muscle cells with thecompound comprises acute, intermittent, or continuous exposure of thepopulation of isolated muscle cells to the compound.

In some embodiments, the population of isolated muscle cells isautologous to the subject. In other embodiments, the population ofisolated muscle cells is allogeneic to the subject.

Administration of the compound can be oral, intraperitoneal,intramuscular, intra-arterial, intradermal, subcutaneous, intravenous,or intracardiac administration. In some cases, the compound isadministered in accordance with an acute regimen. In certain instances,the acute regimen comprises acute exposure (e.g., a single dose) of thecompound to the subject. In other instances, the acute regimen comprisesintermittent exposure (e.g., repeated doses) of the compound to thesubject. As a non-limiting example, an acute PGE2 regimen can comprise aseries of intermittent (e.g., daily) doses of PGE2 over a desired periodof time (e.g., over the course of 2, 3, 4, 5, 6, or 7 days).Administration of the population of isolated muscle cells can includeinjecting or transplanting the cells into the subject. The compound andthe population of isolated muscle cells can be administered to thesubject concomitantly. Optionally, the compound and the population ofisolated muscle cells can be administered to the subject sequentially.

In some embodiments, the subject is suspected of having or at risk fordeveloping the condition or disease associated with muscle damage,injury, or atrophy. In some cases, the condition or disease associatedwith muscle damage, injury or atrophy is selected from the groupconsisting of acute muscle injury or trauma, soft tissue hand injury,Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limbgirdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distalmuscular dystrophy (DD), inherited myopathies, myotonic musculardystrophy (MDD), mitochondrial myopathies, myotubular myopathy (MM),myasthenia gravis (MG), congestive heart failure, periodic paralysis,polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDScachexia, cardiac cachexia, stress induced urinary incontinence, andsarcopenia.

A. Methods for Stimulating the Proliferation or Engraftment of MuscleCells

Provided herein are in vitro or ex vivo methods for stimulating orpromoting the proliferation and/or engraftment of isolated muscle cells.The methods include culturing or contacting a population of isolatedmuscle cells with prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2receptor agonist, a compound that attenuates PGE2 catabolism, a compoundthat neutralizes PGE2 inhibition, a derivative thereof, an analogthereof, or a combination thereof. The compound can be added to anyculture media used to maintain or propagate the cells.

The compound can be any small molecule, prodrug, biological product, andthe like that can mimic, activate, or stimulate PGE2 signaling. In somecases, the compound is PGE2 (i.e., dinoprostone), a synthetic PGE2derivative (e.g., 16,16-dimethyl prostaglandin E2; dmPGE2), a syntheticPGE2 analog, a synthetic PGE2 variant, or a muscle-specific PGE2variant. In other cases, the compound is a PGE2 prodrug such as aprodrug of PGE2 that can be metabolized into a pharmacologically activePGE2 drug when exposed to muscle cells or in close proximity to musclecells. In yet other cases, the compound can be an agonist of any one ofthe PGE2 receptors including PGE2 receptor 1, PGE2 receptor 2, PGE2receptor 3, and PGE2 receptor 4. The agonist can specifically bind to oractivate one or more PGE2 receptors. In some cases, the compound can bea compound that attenuates, impedes, inhibits or decreases PGE2catabolism such as a compound or neutralizing (blocking) antibody thatinactivates or blocks an enzyme that degrades or metabolizes PGE2, e.g.,15-hydroxyprostaglandin dehydrogenase (15-PGDH). In other cases, thecompound blocks, hinders or opposes inhibition of PGE2 and/or PGE2synthesis, activity, and/or secretion.

In some embodiments, the compounds described herein can triggerproliferation of muscle cells including quiescent muscle cells. Thepopulation of isolated muscle cells can be a pure or substantially purepopulation of muscle cells such that at least about 90% of the musclecells are a single type of muscle cell. In other embodiments, thepopulation is a mixture of muscle cells wherein less than about 90% ofthe cells are of one type of cell. In some instances, the muscle cellsinclude skeletal muscle cells, smooth muscle cells, and/or cardiacmuscle cells harvested from a subject. In other instances, the musclecells are generated or differentiated from embryonic stem cells, e.g.,human embryonic stem cells or induced pluripotent stem cells, e.g.,human induced pluripotent stem cells. In yet other instances, the musclecells are dedifferentiated muscle cells. In some embodiments, thepopulation of isolated muscle cells comprises muscle stem cells,satellite cells, myocytes, myoblasts, myotubes, myofibers, or acombination thereof. For instance, the isolated muscle cells can be apure or substantially pure population of muscle stem cells.Alternatively, the isolated muscle cells can be a pure or substantiallypure population of satellite cells. In other instances, the isolatedmuscle cells can a heterogeneous mixture of muscle stem cells, satellitecells, myocytes, myoblasts, myotubes, myofibers, or any combinationthereof. As such, the mixture can include muscle stem cells andsatellite cells, and optionally, myocytes.

In some embodiments, the muscle cells or the induced pluripotent stemcells are derived from a subject with a condition or disease associatedwith muscle damage, injury, or atrophy. In some embodiments, thecondition or disease associated with muscle damage, injury, or atrophyis acute muscle injury, tear or trauma, soft tissue hand injury,Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limbgirdle muscular dystrophy, amyotrophic lateral sclerosis (ALS), distalmuscular dystrophy (DD), inherited myopathies, myotonic musculardystrophy (MDD), mitochondrial myopathies, myotubular myopathy (MM),myasthenia gravis (MG), congestive heart failure, periodic paralysis,polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia, AIDScachexia, cardiac cachexia, stress induced urinary incontinence,sarcopenia or any combination thereof.

In particular embodiments, ex vivo methods for promoting muscle cellengraftment in a subject are provided. The methods include culturing orcontacting a population of isolated muscle cells with an effectiveamount of a compound selected from the group consisting of prostaglandinE2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound thatattenuates PGE2 catabolism, a compound that neutralizes PGE2 inhibition,a derivative thereof, an analog thereof, and a combination thereof, topromote engraftment of the muscle cells in the subject. The methods alsoinclude administering the cultured or contacted muscle cells to thesubject. In some instances, the population of isolated muscle cells isautologous to the subject. In other instances, the population ofisolated muscle cells is allogeneic to the subject. In some embodiments,the subject is a human. In some embodiments, the subject has a conditionor disease associated with muscle damage, injury, or atrophy. In someembodiments, the methods further include administering to the subject atherapeutically effective amount of a compound selected from the groupconsisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptoragonist, a compound that attenuates PGE2 catabolism, a compound thatneutralizes PGE2 inhibition, a derivative thereof, an analog thereof,and a combination thereof, and a pharmaceutically acceptable carrier.The subject can be administered the compound before, simultaneouslywith, and/or after the cultured or contacted muscle cells areadministered to the subject. In some instances, the population ofisolated muscle cells is cultured or contacted with the same compoundthat is administered to the subject. In other instances, the populationof isolated muscle cells is cultured or contacted with a compound thatis different from the compound administered to the subject.

The muscle cells can be obtained from any muscle of the body including,but not limited to, musculi pectoralis complex, latissimus dorsi, teresmajor and subscapularis, brachioradialis, biceps, brachialis, pronatorquadratus, pronator teres, flexor carpi radialis, flexor carpi ulnaris,flexor digitorum superficialis, flexor digitorum profundus, flexorpollicis brevis, opponens pollicis, adductor pollicis, flexor pollicisbrevis, iliopsoas, psoas, rectus abdominis, rectus femoris, gluteusmaximus, gluteus medius, medial hamstrings, gastrocnemius, lateralhamstring, quadriceps mechanism, adductor longus, adductor brevis,adductor magnus, gastrocnemius medial, gastrocnemius lateral, soleus,tibialis posterior, tibialis anterior, flexor digitorum longus, flexordigitorum brevis, flexor hallucis longus, extensor hallucis longus, handmuscles, arm muscles, foot muscles, leg muscles, chest muscles, stomachmuscles, back muscles, buttock muscles, shoulder muscles, head and neckmuscles, and the like.

In some embodiments, the muscle cells are obtained from a particularmuscle, expanded according to the method disclosed herein, and thentransplanted back to the same muscle, or alternatively, transplanted toa different muscle. In some cases, the source of the muscle cells andthe transplantation site is the same muscle of a subject. In othercases, the source of the muscle cells and the transplantation site aredifferent muscles of a subject. In other cases, the source of the musclecells and the transplantation site is the same type of muscle fromdifferent subjects. In yet other cases, the source of the muscle cellsand the transplantation site are different types of muscle fromdifferent subjects.

The compounds disclosed herein can be cultured with isolated musclecells acutely, intermittently or continuously. In some embodiments, thecompound is exposed to the cells in a single dose for a duration oftime. In other embodiments, the compound is exposed to the cells in atleast two or more doses such that a period of time, e.g., a day, twodays, a week or more, passes between dosings. In some embodiments, thecompound is chronically or continuously exposed to the cells, e.g.,without a change in the compound concentration or in the effect on thecells, over a duration of time.

B. Methods for Regenerating Damaged Muscle Cells in a Subject

The methods provided herein can be used to regenerate or rejuvenatemuscle in a subject, such as a human subject. Regeneration of muscleincludes forming new muscle fibers from muscle stem cells, satellitecells, muscle progenitor cells, and any combination thereof. The methodsare also useful for enhancing or augment muscle repair and/ormaintenance.

The PGE2 compounds of the present invention can be administered to asubject experiencing muscle degeneration or atrophy. Muscle atrophy caninclude loss of muscle mass and/or strength. It can affect any muscle ofa subject. In some cases, the subject in need of the compositions,methods, and kits provided herein is exhibiting or experiencing muscleloss due to, e.g., age, inactivity, injury, disease, and any combinationthereof.

In some embodiments, compounds can activate muscle cell proliferation,differentiation, and/or fusion of muscle cells. In some cases, themuscle tissue is regenerated. In other cases, muscle function (e.g.,muscle mass, muscle strength, and/or muscle contraction) is restored orenhanced. In some cases, muscle weakness and atrophy are ameliorated.

The damaged muscle can be any muscle of the body, including but notlimited to, musculi pectoralis complex, latissimus dorsi, teres majorand subscapularis, brachioradialis, biceps, brachialis, pronatorquadratus, pronator teres, flexor carpi radialis, flexor carpi ulnaris,flexor digitorum superficialis, flexor digitorum profundus, flexorpollicis brevis, opponens pollicis, adductor pollicis, flexor pollicisbrevis, iliopsoas, psoas, rectus abdominis, rectus femoris, gluteusmaximus, gluteus medius, medial hamstrings, gastrocnemius, lateralhamstring, quadriceps mechanism, adductor longus, adductor brevis,adductor magnus, gastrocnemius medial, gastrocnemius lateral, soleus,tibialis posterior, tibialis anterior, flexor digitorum longus, flexordigitorum brevis, flexor hallucis longus, extensor hallucis longus, handmuscles, arm muscles, foot muscles, leg muscles, chest muscles, stomachmuscles, back muscles, buttock muscles, shoulder muscles, head and neckmuscles, facial muscles, oculopharyngeal muscles, and the like.

Subjects in need of muscle regeneration may have musculoskeletalinjuries (e.g., fractures, strains, sprains, acute injuries, overuseinjuries, and the like), post-trauma damages to limbs or face, athleticinjuries, post-fractures in the aged, soft tissue hand injuries, muscleatrophy (e.g., loss of muscle mass), Duchenne muscular dystrophy (DMD),Becker muscular dystrophy, Fukuyama congenital muscular dystrophy(FCMD), limb-girdle muscular dystrophy (LGMD), congenital musculardystrophy, facioscapulohumeral muscular dystrophy (FHMD), myotonicmuscular dystrophy, oculopharyngeal muscular dystrophy, distal musculardystrophy, Emery-Dreifuss muscular dystrophy, myotonia congenita,myotonic dystrophy, other muscular dystrophies, muscle wasting disease,such as cachexia due to cancer, end stage renal disease (ESRD), acquiredimmune deficiency syndrome (AIDS), or chronic obstructive pulmonarydisease (COPD), post-surgical muscle weakness, post-traumatic muscleweakness, sarcopenia, inactivity (e.g., muscle disuse or immobility),urethral sphincter deficiency, urethral sphincter deficiency,neuromuscular disease, and the like.

Non-limiting examples of neuromuscular diseases include, but are notlimited to, acid maltase deficiency, amyotrophic lateral sclerosis,Andersen-Tawil syndrome, Becker muscular dystrophy, Becker myotoniacongenita, Bethlem myopathy, bulbospinal muscular atrophy, camitinedeficiency, camitine palmityl transferase deficiency, central coredisease, centronuclear myopathy, Charcot-Marie-Tooth disease, congenitalmuscular dystrophy, congenital myasthenic syndromes, congenital myotonicdystrophy, Cori disease, Debrancher enzyme deficiency, Dejerine-Sottasdisease, dermatomyositis, distal muscular dystrophy, Duchenne musculardystrophy, dystrophia myotonica, Emery-Dreifuss muscular dystrophy,endocrine myopathies, Eulenberg disease, facioscapulohumeral musculardystrophy, tibial distal myopathy, Friedreich's ataxia, Fukuyumacongenital muscular dystrophy, glycogenosis type 10, glycogenosis type11, glycogenosis type 2, glycogenosis type 3, glycogenosis type 5,glycogenosis type 7, glycogenosis type 9, Gowers-Laing distal myopathy,hereditary inclusion-body myositis, hyperthyroid myopathy, hypothyroidmyopathy, inclusion-body myositis, inherited myopathies,integrin-deficient congenital muscular dystrophy, spinal-bulbar muscularatrophy, spinal muscular atrophy, lactate dehydrogenase deficiency,Lambert-Eaton myasthenic syndrome, McArdel disease, merosin-deficientcongenital muscular dystrophy, metabolic diseases of muscle,mitochondrial myopathy, Miyoshi distal myopathy, motor neuron disease,muscle-eye-brain disease, myasthenia gravis, myoadenylate deaminasedeficiency, myofibrillar myopathy, myophosphorylase deficiency, myotoniacongenital, myotonic muscular dystrophy, myotubular myopathy, nemalinemyopathy, Nonaka distal myopathy, oculopharyngeal muscular dystrophy,paramyotonia congenital, Pearson syndrome, periodic paralysis,phosphofructokinase deficiency, phosphoglycerate kinase deficiency,phosphoglycerate mutase deficiency, phosphorylase deficiency,polymyositis, Pompe disease, progressive external ophthalmoplegia,spinal muscular atrophy, Ullrich congenital muscular dystrophy, Welanderdistal myopathy, ZASP-related myopathy, and the like.

Muscle atrophy (e.g., muscle wasting) can be caused by or associatedwith, for example, normal aging (e.g., sarcopenia), geneticabnormalities (e.g., mutations or single nucleotide polymorphisms), poornourishment, poor circulation, loss of hormonal support, disuse of themuscle due to lack of exercise (e.g., bedrest, immobilization of a limbin a cast, etc.), aging, damage to the nerve innervating the muscle,poliomyelitis, amyotrophic lateral sclerosis (ALS or Lou Gehrig'sdisease), heart failure, liver disease, diabetes, obesity, metabolicsyndrome, demyelinating diseases (e.g., multiple sclerosis,Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher disease,encephalomyelitis, neuromyelitis optica, adrenoleukodystrophy, andGuillian-Barre syndrome), denervation, fatigue, exercise-induced musclefatigue, frailty, neuromuscular disease, weakness, chronic pain, and thelike.

In some aspects, provided herein are methods for regenerating muscle ina subject in need thereof by administering to the subject atherapeutically effective amount of a compound selected from the groupconsisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptoragonist, a compound that attenuates PGE2 catabolism, a compound thatneutralizes PGE2 inhibition, a derivative thereof, an analog thereof,and a combination thereof, and a pharmaceutically acceptable carrier, toincrease the population of muscle cells and/or to enhance musclefunction in the subject. The population of muscle cells in the subjectcan include skeletal muscle cells, smooth muscle cells, cardiac musclecells, embryonic stem cell-derived muscle cells, induced pluripotentstem cell-derived muscle cells, dedifferentiated muscle cells, or anycombinations thereof. Additionally, the muscle cells in the subject canbe muscle stem cells, satellite cells, myocytes, myoblasts, myotubes,myofibers, or any combination thereof. The compound can be administeredto the subject by oral, intraperitoneal, intramuscular, intra-arterial,intradermal, subcutaneous, intravenous, or intracardiac administration.In some cases, the compound is administered directly to thedysfunctional, injured, damaged and/or atrophied muscle. The compoundcan be administered in accordance with an acute regimen (e.g., single orintermittent dosing) or a chronic regimen (e.g., continuous dosing).

In some embodiments, the subject is also administered a population ofisolated (or isolated and purified) muscle cells that are eitherautologous or allogeneic to the subject. The cells can be isolatedand/or purified by any method known to those of skill in the art. Thecells can be a homogenous or heterogeneous population of muscle cells.

In some embodiments, the cells are stimulated to proliferate byculturing the cells with the PGE2 compound prior to administering themto the subject. The cells can be acutely, intermittently or continuouslyexposed to the compound during in vitro culturing. In some cases, thepopulation of muscle cells increases by at least about 1%, at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about100%, at least about 200%, at least about 500%, at least about 1000%, ormore after culturing with the PGE2 compound.

To regenerate or repair muscle in the subject, the compound of thepresent invention and the isolated muscle cells are administered to thesubject concomitantly. In some embodiments, the compound and thecultured muscle cells are administered to the subject concomitantly. Inother embodiments, the compound and the isolated muscle cells areadministered to the subject sequentially. In yet other embodiments, thecompound and the cultured muscle cells are administered to the subjectsequentially.

The methods described herein can be used to increase the number ofmuscle fibers by at least about 1%, at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 100%, at least about200%, at least about 500%, at least about 1000%, or more. In someembodiments, the methods can increase the growth of a damaged, injured,atrophied, or degenerated muscle.

C. Methods for Preventing or Treating a Condition or Disease AffectingMuscle

The methods provided herein can be used to prevent or treat a conditionor disease associated with muscle damage, injury, or atrophy in asubject in need thereof. The method can provide prophylactic treatmentto a subject who is likely to experience muscle damage, injury oratrophy. In some embodiments, the subject can have a condition ordisease with possible secondary symptoms that affect muscle. In otherembodiments, the subject has undergone a surgical or therapeuticintervention to treat the muscle condition or disease, and the methoddisclosed here is used to prevent or inhibit recurrence or relapse. Insome embodiments, the subject has any one of the conditions or diseasesdescribed herein that affects muscle.

As used herein, the term “treatment” or “treating” encompassesadministration of compounds and/or cells in an appropriate form prior tothe onset of disease symptoms and/or after clinical manifestations, orother manifestations of the condition or disease to reduce diseaseseverity, halt disease progression, or eliminate the disease. The term“prevention of” or “preventing” a disease includes prolonging ordelaying the onset of symptoms of the condition or disease, preferablyin a subject with increased susceptibility to the condition or disease.

The method includes administering to the subject (i) a therapeuticallyeffective amount of a compound selected from the group consisting ofprostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, acompound that attenuates PGE2 catabolism, a compound that neutralizesPGE2 inhibition, a derivative thereof, an analog thereof, and acombination thereof, and a pharmaceutically acceptable carrier, and (ii)a population of isolated muscle cells, to prevent or treat the conditionor disease associated with muscle damage, injury, or atrophy. The musclecells can be autologous or allogeneic to the subject.

The compound can be administered orally, intraperitoneally,intramuscularly, intra-arterially, intradermally, subcutaneously,intravenously, or by intracardiac injection. The compound can beadministered in accordance with an acute regimen (e.g., single orintermittent dosing) or a chronic regimen (e.g., continuous dosing). Theisolated muscle cells can be administered by injection ortransplantation. In some embodiments, the compound and the cells areadministered together or concomitantly. In other embodiments, thecompound and the cells are administered sequentially. In some cases, thecompound is administered before the cells. In other cases, the cells areadministered before the compound.

The isolated muscle cells can be substantially purified or purifiedprior to injection or transplantation into the subject. The cells canalso be expanded or stimulated to proliferate in culture prior toadministration. As described herein, isolated muscle cells includingskeletal muscle cells, smooth muscle cells, cardiac muscle cells,embryonic stem cell-derived muscle cells, induced pluripotent stemcell-derived muscle cells, dedifferentiated muscle cells, muscle stemcells, satellite cells, myoblasts, myocytes, myotubes, myofibers, andany combination thereof can be cultured with the compounds of thepresent invention. By exposing the compound to the cells acutely,intermittently or continuously, the muscle cells proliferate andincrease in number. The expanded cells can be transplanted into asubject experiencing muscle damage, injury or atrophy.

D. Prostaglandin E2 (PGE2) Compounds

In some embodiments, the compound of the present invention is selectedfrom the group consisting of PGE2, a PGE2 prodrug, a PGE2 receptoragonist, a compound that attenuates PGE2 catabolism, a compound thatneutralizes PGE2 inhibition, a derivative thereof, an analog thereof,and a combination thereof. In some cases, a compound that attenuatesPGE2 catabolism can be a compound, a neutralizing peptide, or aneutralizing antibody that inactivates or blocks 15-hydroxyprostaglandindehydrogenase (15-PGDH) or inactivates or blocks a prostaglandintransporter, which transports PGE2 inside cells for catabolism by15-PGDH. The prostaglandin transporter is also known as 2310021C19Rik,MATRI, Matrin F/Q, OATP2A1, PGT, PHOAR2, SLC21A2, solute carrier organicanion transporter family member 2A1, and SLCO2A1.

The PGE2 receptor agonist can be a small molecule compound, anactivating antibody that specifically binds to a PGE2 receptor, and thelike. In some embodiments, the compound is a PGE2 derivative or analog.In some embodiments, the compound is a PGE2 prodrug. A prodrug of PGE2can be metabolized into a pharmacologically active PGE2 drug, forexample, at the site of administration or muscle regeneration, or whenthe prodrug is exposed to muscle cells.

In particular embodiments, the compound is a PGE2 derivative or analogthat contains one or more modifications to PGE2 that increase itsstability, activity, resistance to degradation, transport into musclecells (e.g., promote cellular uptake), and/or retention in muscle cells(e.g., reduce secretion from muscle cells after uptake).

Without limitation, examples of PGE2 derivatives and analogs include2,2-difluoro-16-phenoxy-PGE2 compounds,2-decarboxy-2-hydroxymethyl-16-fluoro-PGE2 compounds,2-decarboxy-2-hydroxymethyl-1-deoxy-PGE2 compounds, 19(R)-hydroxy PGE2,16,16-dimethyl PGE2, 16,16-dimethyl PGE2 p-(p-acetamidobenzamido) phenylester, 11-deoxy-16,16-dimethyl PGE2, 9-deoxy-9-methylene-16,16-dimethylPGE2, 9-deoxy-9-methylene PGE2, butaprost, sulprostone, enprostil, PGE2serinol amide, PGE2 methyl ester, 16-phenyl tetranor PGE2, 5-trans-PGE2,15(S)-15-methyl PGE2, and 15(R)-15-methyl PGE2. Additional PGE2derivatives and analogs are set forth, e.g., in U.S. Pat. No. 5,409,911.

Additional non-limiting examples of PGE2 derivatives and analogs includehydantoin derivatives of PGE2, the more stable PGE2 analogs described inZhao et al. (Bioorganic & Medicinal Chemistry Letters, 17:6572-5 (2007))in which the hydroxy cyclopentanone ring is replaced by heterocyclicrings and the unsaturated alpha-alkenyl chain is substituted with aphenethyl chain, the PGE2 analogs described in Ungrin et al. (Mol.Pharmacol., 59:1446-56 (2001)), the 13-dehydro derivatives of PGE2described in Tanami et al. (Bioorg. Med. Chem. Lett., 8:1507-10 (1998)),and the substituted cyclopentanes described in U.S. Pat. Nos. 8,546,603and 8,158,676.

In some embodiments, the compound is an agonist of a PGE2 receptor,e.g., EP1 receptor, EP2 receptor, EP3 receptor, and EP4 receptor.Non-limiting examples of PGE2 receptor agonists include ONO-DI-004,ONO-AEI-259, ONO-AE-248, ONO-AE1-329, ONO-4819CD (Ono PharmaceuticalCo., Japan), L-902688 (Cayman Chemical), CAY10598 (Cayman Chemical), andCP-533536 (Pfizer). Additional PGE2 receptor agonists are described,e.g., in U.S. Pat. Nos. 6,410,591; 6,610,719; 6,747,037; 7,696,235;7,662,839; 7,652,063; 7,622,475; and 7,608,637.

E. Isolated Muscle Cells

Muscle (myogenic) cells of the present invention include, but are notlimited to, muscle stem cells, skeletal muscle stem cells, smooth musclestem cells, cardiac muscle stem cells, muscle satellite cells, myogenicprecursor cells, myogenic cells, myocytes, myoblasts, myotubes,postmitotic myotubes, multinucleated myofibers, and postmitotic musclefibers. In some embodiments, the isolated muscle cells encompass musclestem cells. In other embodiments, the isolated muscle cells includemuscle satellite cells. The muscle cells can be derived from a stem cellsuch as a bone marrow-derived stem cell, or a pluripotent stem cell suchas an embryonic stem cell or an induced pluripotent stem cell. In someembodiments, the isolated muscle cells include dedifferentiated musclecells. In other embodiments, the muscle cells have been geneticallymodified to, in some cases, correct disease-associated gene mutations.

Satellite cells are small mononuclear progenitor cells that can residewithin muscle tissue. These cells can be induced to proliferate anddifferentiate into muscle cells, and in some instances, fuse to musclefibers. During muscle damage or injury, quiescent satellite cells (e.g.,satellite cells that are not differentiating or undergoing cell divisionat present) and muscle stem cells can be activated to proliferate,and/or migrate out of the muscle stem cell niche. The satellite cellsand muscle stem cells can also differentiate into myocytes, myoblasts,or other muscle cell types.

Methods and protocols for generating muscle cells from embryonic stemcells are described, e.g., in Hwang et al., PLoS One, 2013, 8(8):e72023;and Darabi et al., Cell Stem Cell, 2012, 10(5):610-9. Methods andprotocols for generating muscle cells from induced pluripotent stemcells are described, e.g., in Darabi et al., Cell Stem Cell, 2012,10(5):610-9; Tan et al., PLoS One, 2011; and Mizuno et al., FASEB J.,2010, 24(7):2245-2253.

In some embodiments, muscle cells are obtained by biopsy from a musclesuch as a mature or adult muscle, e.g., quadriceps, gluteus maximus,bicep, triceps, or any muscle from an individual. The muscle can be askeletal muscle, smooth muscle, or cardiac muscle. Detailed descriptionsof methods of isolating smooth muscle stem cells can be found, e.g., inU.S. Pat. No. 8,747,838, and U.S. Patent App. Publ. No. 20070224167.Methods of isolating muscle cells of interest such as muscle stem cellsor satellite cells from muscle tissue are described in detail, forexample, in Blanco-Bose et al., Exp. Cell Res., 2001, 26592:212-220.

Methods for purifying a population of muscle cells of interest, e.g.,muscle stem cells, muscle satellite cells, myocytes, myoblasts,myotubes, and/or myofibers include selecting, isolating or enriching fora cell having a specific cell surface marker or a specific polypeptidethat is expressed on the cell surface of the muscle cell of interest.Useful cell surface markers are described in, e.g., Fukada et al.,Front. Physiol., 2013, 4:317. Cell sorting methods such as flowcytometry, e.g., fluorescence-activated cell sorting (FACS); magneticbead cell separation, e.g., magnetic-activated cell sorting (MACS), andother antibody-based cell sorting methods can be performed to isolate orseparate the muscle cells of interest from other cell types.

The isolated population of muscle cells of interest can be expanded ormultiplied using conventional culture-based methods. Methods for culturemuscle cells are found in, e.g., U.S. Pat. No. 5,324,656. In some cases,the cells are cultured on a scaffold or gel such as a hydrogel.

F. Methods of Administration

The compounds of the present invention can be administered locally at ornear a site of injury in the subject or systemically. In someembodiments, the compounds can be administered, for example,intraperitoneally, intramuscularly, intra-arterially, orally,intravenously, intracranially, intrathecally, intraspinally,intralesionally, intranasally, subcutaneously,intracerebroventricularly, topically, and/or by inhalation. The compoundmay be administered simultaneously or sequentially with the muscle cellsof interest. When the compound is administered simultaneously with thecells, both the compound and cells can be administered in the samecomposition. When administered separately, the compound can be providedin a pharmaceutically acceptable carrier. In some embodiments, thecompound is administered before or after the administration of thecells.

In some embodiments, the compound is administered in accordance with anacute regimen. In certain instances, the compound is administered to thesubject once. In other instances, the compound is administered at onetime point, and administered again at a second time point. In yet otherinstances, the compound is administered to the subject repeatedly (e.g.,once or twice daily) as intermittent doses over a short period of time(e.g., 2 days, 3 days, 4 days, 5 days, 6 days, a week, 2 weeks, 3 weeks,4 weeks, a month, or more). In some cases, the time between compoundadministrations is about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days,a week, 2 weeks, 3 weeks, 4 weeks, a month, or more. In otherembodiments, the compound is administered continuously or chronically inaccordance with a chronic regimen over a desired period of time. Forinstance, the compound can be administered such that the amount or levelof the compound is substantially constant over a selected time period.

Administration of the isolated muscle cells into a subject can beaccomplished by methods generally used in the art. In some embodiments,administration is by transplantation or injection such as intramuscularinjection. The number of cells introduced will take into considerationfactors such as sex, age, weight, the types of disease or disorder,stage of the disorder, the percentage of the desired cells in the cellpopulation (e.g., purity of cell population), and the cell number neededto produce the desired result. Generally, for administering the cellsfor therapeutic purposes, the cells are given at a pharmacologicallyeffective dose. By “pharmacologically effective amount” or“pharmacologically effective dose” is an amount sufficient to producethe desired physiological effect or amount capable of achieving thedesired result, particularly for treating the condition or disease,including reducing or eliminating one or more symptoms or manifestationsof the condition or disease. Pharmacologically effective doses will alsoapply to therapeutic compounds used in combination with the cells, asdescribed herein.

Cells can be administered in one injection, or through successiveinjections over a defined time period sufficient to generate atherapeutic effect. Different populations of muscle cells may beinjected when treatment involves successive injections. Apharmaceutically acceptable carrier, as further described below, may beused for injection of the cells into the subject. These will typicallycomprise, for example, buffered saline (e.g., phosphate buffered saline)or unsupplemented basal cell culture medium, or medium as known in theart.

Any number of muscles of the body may be directly injected with thecompound and/or cells of the present invention, such as, for example,the biceps muscle; the triceps muscle; the brachioradialus muscle; thebrachialis muscle (brachialis anticus); the superficial compartmentwrist flexors; the deltoid muscle; the biceps femoris, the gracilis, thesemitendinosus and the semimembranosus muscles of the hamstrings; therectus femoris, vastus lateralis, vastus medialis and vastus intermediusmuscles of the quadriceps; the gastrocnemius (lateral and medial),tibialis anterior, and the soleus muscles of the calves; the pectoralismajor and the pectoralis minor muscles of the chest; the latissimusdorsi muscle of the upper back; the rhomboids (major and minor); thetrapezius muscles that span the neck, shoulders and back; the rectusabdominis muscles of the abdomen; and the gluteus maximus, gluteusmedius and gluteus minimus muscles of the buttocks.

G. Pharmaceutical Compositions

The pharmaceutical compositions of the compounds and cells of thepresent invention may comprise a pharmaceutically acceptable carrier. Incertain aspects, pharmaceutically acceptable carriers are determined inpart by the particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see e.g., REMINGTON'SPHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, Pa.(1990)).

As used herein, “pharmaceutically acceptable carrier” comprises any ofstandard pharmaceutically accepted carriers known to those of ordinaryskill in the art in formulating pharmaceutical compositions. Thus, thecells or compounds, by themselves, such as being present aspharmaceutically acceptable salts, or as conjugates, may be prepared asformulations in pharmaceutically acceptable diluents; for example,saline, phosphate buffer saline (PBS), aqueous ethanol, or solutions ofglucose, mannitol, dextran, propylene glycol, oils (e.g., vegetableoils, animal oils, synthetic oils, etc.), microcrystalline cellulose,carboxymethyl cellulose, hydroxylpropyl methyl cellulose, magnesiumstearate, calcium phosphate, gelatin, polysorbate 80 or the like, or assolid formulations in appropriate excipients.

The pharmaceutical compositions will often further comprise one or morebuffers (e.g., neutral buffered saline or phosphate buffered saline),carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol,proteins, polypeptides or amino acids such as glycine, antioxidants(e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene,butylated hydroxyanisole, etc.), bacteriostats, chelating agents such asEDTA or glutathione, solutes that render the formulation isotonic,hypotonic or weakly hypertonic with the blood of a recipient, suspendingagents, thickening agents, preservatives, flavoring agents, sweeteningagents, and coloring compounds as appropriate.

The pharmaceutical compositions of the invention are administered in amanner compatible with the dosage formulation, and in such amount aswill be therapeutically effective. The quantity to be administereddepends on a variety of factors including, e.g., the age, body weight,physical activity, and diet of the individual, the condition or diseaseto be treated, and the stage or severity of the condition or disease. Incertain embodiments, the size of the dose may also be determined by theexistence, nature, and extent of any adverse side effects that accompanythe administration of a therapeutic agent(s) in a particular individual.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, hereditary characteristics, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and the hostundergoing therapy.

In certain embodiments, the dose of the compound may take the form ofsolid, semi-solid, lyophilized powder, or liquid dosage forms, such as,for example, tablets, pills, pellets, capsules, powders, solutions,suspensions, emulsions, suppositories, retention enemas, creams,ointments, lotions, gels, aerosols, foams, or the like, preferably inunit dosage forms suitable for simple administration of precise dosages.

As used herein, the term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosages for humans and other mammals,each unit containing a predetermined quantity of a therapeutic agentcalculated to produce the desired onset, tolerability, and/ortherapeutic effects, in association with a suitable pharmaceuticalexcipient (e.g., an ampoule). In addition, more concentrated dosageforms may be prepared, from which the more dilute unit dosage forms maythen be produced. The more concentrated dosage forms thus will containsubstantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more times the amount of the therapeutic compound.

Methods for preparing such dosage forms are known to those skilled inthe art (see. e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Thedosage forms typically include a conventional pharmaceutical carrier orexcipient and may additionally include other medicinal agents, carriers,adjuvants, diluents, tissue permeation enhancers, solubilizers, and thelike. Appropriate excipients can be tailored to the particular dosageform and route of administration by methods well known in the art (see,e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra).

Examples of suitable excipients include, but are not limited to,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,saline, syrup, methylcellulose, ethylcellulose,hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols,e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The dosage formscan additionally include lubricating agents such as talc, magnesiumstearate, and mineral oil; wetting agents; emulsifying agents;suspending agents; preserving agents such as methyl-, ethyl-, andpropyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents suchas inorganic and organic acids and bases; sweetening agents; andflavoring agents. The dosage forms may also comprise biodegradablepolymer beads, dextran, and cyclodextrin inclusion complexes.

For oral administration, the therapeutically effective dose can be inthe form of tablets, capsules, emulsions, suspensions, solutions,syrups, sprays, lozenges, powders, and sustained-release formulations.Suitable excipients for oral administration include pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesiumcarbonate, and the like.

The therapeutically effective dose can also be provided in a lyophilizedform. Such dosage forms may include a buffer, e.g., bicarbonate, forreconstitution prior to administration, or the buffer may be included inthe lyophilized dosage form for reconstitution with, e.g., water. Thelyophilized dosage form may further comprise a suitable vasoconstrictor,e.g., epinephrine. The lyophilized dosage form can be provided in asyringe, optionally packaged in combination with the buffer forreconstitution, such that the reconstituted dosage form can beimmediately administered to an individual.

H. Kits

Other embodiments of the compositions described herein are kitscomprising a population of isolated muscle cells and a compound selectedfrom the group consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, aPGE2 receptor agonist, a compound that attenuates PGE2 catabolism (e.g.,15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitor orprostaglandin transporter (PTG or SLCO2A1) inhibitor), a compound thatneutralizes PGE2 inhibition, a derivative thereof, an analog thereof,and a combination thereof. The kit typically contains containers whichmay be formed from a variety of materials such as glass or plastic, andcan include for example, bottles, vials, syringes, and test tubes. Alabel typically accompanies the kit, and includes any writing orrecorded material, which may be electronic or computer readable formproviding instructions or other information for use of the kit contents.

V. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1: Acute Prostaglandin E2 Delivery Augments Skeletal MuscleRegeneration and Strength in Aged Mice

This example illustrates that PGE2 signaling is required for muscle stemcell function during regeneration.

The elderly suffer from progressive skeletal muscle wasting andregenerative failure that decreases mobility and quality of life^(1,2).Crucial to muscle regeneration are adult muscle stem cells (MuSCs) thatreside in niches in muscle tissues, poised to respond to damage andrepair skeletal muscles throughout life³⁻⁸. During aging, the proportionof functional MuSCs markedly decreases, hindering muscleregeneration⁹⁻³. To date, no therapeutic agents are in clinical use thattarget MuSCs to combat this regenerative decline. Here, we identify anatural immunomodulator, prostaglandin E2 (PGE2), as a potent regulatorof MuSC function essential to muscle regeneration. We found that thePGE2 receptor, EP4, is essential for MuSC proliferation in vitro andengraftment in vivo in mice. In MuSCs of aged mice, the PGE2 pathway isdysregulated due to a cell intrinsic molecular defect, elevatedprostaglandin degrading enzyme (15-PGDH) that renders PGE2 inactive.This defect is overcome by transient acute exposure of MuSCs to a stabledegradation-resistant PGE2, 16,16-dimethyl PGE2 (dmPGE2), concomitantwith MuSC transplantation into injured muscles. Notably, a singleintramuscular injection of dmPGE2 alone suffices to accelerateregeneration, evident by an early increase in endogenous MuSC numbersand myofiber sizes following injury. Furthermore, aged mouse muscleforce generating capacity was increased in response to exercise-inducedregeneration and an acute dmPGE2 treatment regimen. Our findings reveala novel therapeutic indication for PGE2 as a potent inducer of muscleregeneration and strength.

To counter the decline in muscle regenerative potential we soughttherapeutic agents that target MuSCs, also known as satellite cells, astem cell population dedicated to muscle regeneration³⁻⁸. Since atransient inflammatory and fibroadipogenic response plays a crucial rolein muscle regeneration¹⁴⁻¹⁷, we sought to identify inflammatorymodulators induced by injury that could overcome the age-related declinein MuSC function. An analysis of our transcriptome database revealedthat the Ptger4 receptor for PGE2, a natural and potent lipid mediatorduring acute inflammation¹⁸, was expressed at high levels on freshlyisolated MuSCs. In muscle tissue lysates, we detected a surge in levelsof PGE2 three days after injury to young (2-4 mo) mouse muscles bystandard injury paradigms entailing notexin injection or cryoinjury(FIG. 1A and FIG. 5A), and a concomitant upregulation of itssynthesizing enzymes, Ptges and Ptges2 (FIG. 1B). This early andtransient time window coincides with the well-documented kinetics ofMuSC expansion and inflammatory cytokine accumulation postinjury^(8,15,16). To determine if PGE2 treatment enhanced MuSC behavior,we FACS-purified MuSCs from hindlimb muscles from young mice (2-4 mo)⁶and plated them on hydrogels of 12 kpa stiffness to maintain stem cellfunction¹⁹. We found that PGE2 (10 ng/ml) increased cell divisionassayed by EDU incorporation (FIGS. 1B-ID) and that an acute 1-dayexposure to PGE2 induced a 6-fold increase in the number of MuSCsrelative to controls one week later (FIG. 1C).

PGE2 is known to signal through four G-protein coupled receptors(Ptger1-4; EP1-4)^(18,20), but the expression of these receptors inMuSCs has not previously been described. An analysis of the transcriptlevels of the different receptors (Ptger1-4) revealed that the onlyreceptors upregulated after PGE2 treatment of MuSCs are Ptger1 andPtger4 (FIG. 5E). PGE2 stimulated MuSCs had elevated intracellularcAMP^(18,20) confirming that PGE2 signals through EP4 to promoteproliferation and a stem cell transcriptional state (FIGS. 5F-5H). Inthe presence of an EP4 antagonist, ONO-AE3-208, proliferation induced byPGE2 was blunted (FIG. 1D). However, the specificity of PGE2 for EP4 wasmost clearly shown in MuSCs lacking the receptor following cre-mediatedconditional ablation (FIGS. 1E-1G and FIGS. 5I-5J). Indeed, even in thepresence of growth factor-rich media, these EP4-null MuSCs failed toproliferate. Finally, we found that MuSCs growth arrested by exposure tomedium with charcoal stripped serum²¹, divided upon addition of PGE2(FIG. 1H and FIG. 5K). Thus, PGE2/EP4 stands out as necessary andsufficient for MuSC proliferation.

We sought to determine if PGE2 could ameliorate the muscle regenerativedefects previously reported for aged MuSCs⁹⁻¹³. By contrast with youngmouse muscles (2-4 mo), notexin damage to aged muscles (18-20 mo) didnot lead to an increase in PGE2 synthesis. Instead, steady state PGE2levels in aged muscle remained unchanged post injury (FIG. 2A) and weresignificantly higher than in young limb tibialis anterior (TA) muscles(FIG. 2B). We hypothesized that the PGE2 in aged muscle might bedysfunctional due to a catabolic defect. Indeed, when we analyzed thePGE2 present in young and aged TA muscle tissues by mass spectrometry,we found that the relative amount of the inactive form,13,14-dihydro-15-keto PGE2 (PGEM), was significantly increased in theaged (FIGS. 2C-2D and FIGS. 6A-6C). This proved to be due to aconcomitant 7-fold increase in levels of mRNA encoding the PGE2degrading enzyme (15-PGDH), the initial step in the conversion of PGE2to its inactive form (FIG. 2E). In contrast, the relative levels of theprostaglandin transporter (PGT), PGE2 synthesizing enzymes, and EP4receptor did not differ between young and aged MuSCs (FIGS. 7A-7C).Additionally, when aged MuSCs were exposed to a 1-day pulse of PGE2 orto an inhibitor of 15-PGDH (SW033291)²², the effects of 15-PGDH wereovercome and the characteristic increase in proliferation andmaintenance of Pax7 expression was observed (FIG. 2F and FIG. 7D). Likeyoung, aged MuSCs failed to proliferate in medium comprised of charcoalstripped serum, but were rescued by addition of PGE2 alone (FIG. 2G). Wesurmised that in aged MuSCs the PGE2 pathway is dysregulated due to acell intrinsic molecular defect, elevated 15-PGDH that can be surmountedin culture by acute exposure to PGE2 or SW (FIG. 2H).

Since aged MuSCs are heterogeneous¹⁰, we sought to determine the effectof PGE2 at the single cell level. Clonal analysis can reveal differencesthat are masked by analysis of the population as a whole. Accordingly,we performed long-term time-lapse microscopy in hydrogel ‘microwells’ ofsingle aged MuSCs transiently exposed to PGE2 for 1 day and untreatedcontrol MuSCs. Data were collected over a 48 h time period and thenanalyzed using our previously described Baxter Algorithms for CellTracking and Lineage Reconstruction^(10,19,23). We observed a remarkableincrease in cumulative cell numbers in response to PGE2, spanning 6generations for the most robust clones (FIGS. 2I-2J). The numbers ofcells per clone following PGE2 treatment were significantly augmenteddue to a marked increase in proliferation (FIGS. 21-2J and FIGS. 7E-7F)that was accompanied by a profound reduction in cell death (FIG. 2J andFIGS. 7E-7G). These synergistic effects led to the observed increases inaged MuSC numbers in response to PGE2.

To test whether transient treatment of young MuSCs with PGE2 augmentsregeneration, we transplanted cultured PGE2 treated MuSCs into injuredhindlimb muscles of mice. To monitor the dynamics of regeneration overtime in a quantitative manner in vivo, we capitalized on a sensitive andquantitative bioluminescence imaging (BLI) assay we previously developedfor monitoring MuSC function post-transplantation^(6,10,19). MuSCs wereisolated from young transgenic mice (2-4 mo) expressing GFP andluciferase (GFP/Luc mice), exposed to an acute 1-day PGE2 treatment,harvested and transplanted on day 7. Equivalent numbers of dmPGE2treated and control MuSCs (250 cells) were transplanted into injuredhindlimbs of young (2-4 mo) NOD-SCID mice. Following acute treatmentwith PGE2, young MuSC regenerative capacity was enhanced by an order ofmagnitude when assessed by BLI (FIG. 3A). In contrast, followingtransplantation of 4-fold greater numbers of cultured MuSCs that lackedthe EP4 receptor due to conditional ablation (FIG. 3B), the BLI signalthat was initially detected progressively declined to levels below thethreshold of significance (FIG. 3B).

Furthermore, when notexin injury was performed in the mouse model ofmuscle stem cell specific deletion of EP4 (Pax7^(CreET2);ErEP4^(fl/fl))(FIGS. 9A-9B), muscle regeneration was impaired as observed by theelevated number of embryonic myosin heavy chain (eMHC) positive fibers(FIGS. 9C-9D). This was accompanied by the reduction in cross-sectionalarea of the mouse fibers in the Pax7^(CreERT2):EP4^(fl/f) group,assessed at the end of the regeneration time point (day 21) (FIG. 9E). Asignificant reduction in force output (tetanus) was also detected at day14 post-injury (FIGS. 9F-9G). Thus, PGE2 signaling via the EP4 receptoris required for MuSC regeneration in vivo.

To test if direct injection of PGE2 without culture could be effectivein promoting regeneration in vivo, we coinjected PGE2 together withfreshly isolated MuSCs. For all subsequent in vivo injectionexperiments, we used a modified, more stable form of PGE2,16,16-dimethyl PGE2 (dmPGE2)²⁴. We hypothesized that for the aged MuSCexperiments, the delivery of the modified 15-PGDH-resistant dmPGE2 wasparticularly important, as 15-PGDH is significantly elevated in agedMuSCs (FIG. 2E)²⁴. Using dmPGE2, we observed significantly enhancedengraftment of young and aged MuSCs relative to controls that wasfurther increased in response to notexin injury, a well-acceptedstringent test of stem cell function (FIGS. 3C-3D). Thus, the deliveryof dmPGE2 together with MuSC cell populations suffices to augmentregeneration.

We postulated that delivery of PGE2 alone could stimulate muscleregeneration. To test this, muscles of young mice were injured withcardiotoxin and three days later a bolus of dmPGE2 was injected into thehindlimb muscles of young mice. We observed an increase (60+15%) inendogenous PAX7-expressing MuSCs in the classic satellite cell nichebeneath the basal lamina and atop myofibers fourteen days post injury(FIGS. 4A-4B), whereas dmPGE2 had no effect in the absence of injury.Further, at this early time point, the distribution of myofibers shiftedtoward larger sizes, assessed as cross-sectional area using the BaxterAlgorithms for Myofiber Analysis, suggesting that regeneration isaccelerated by PGE2 (FIGS. 4C-4D and FIGS. 8A-8B). In addition, wetracked the response to injury and dmPGE2 of endogenous MuSCs byluciferase expression using a transgenic mouse model, Pax7^(creERT2);Rosa26-LSL-Luc (FIG. 4E). The BLI data were in agreement with thehistological data (FIGS. 4F-4G).

We tested the effects of injecting indomethacin, a nonsteroidalanti-inflammatory drug (NSAID) and an inhibitor of COX2 which reducesPGE2 synthesis, on muscle regeneration. Upon indomethacin injection intothe hindlimb muscles of the same Pax7^(creERT2);Rosa26-LSL-Luc mousemodel three days post-cardiotoxin injury, we observed a significantdecrease in luciferase activity indicative of an impairment in musclestem cell activation and regeneration (FIGS. 10A-10B). Injection ofindomethacin into cardiotoxin-injured muscles also led to a significantloss in Twitch force as compared to the control group assessed at day 14post-injury (FIG. 10C). In aged mice, we also detected a substantialincrease (24±2%) in the number of endogenous MuSCs (FIGS. 4H-4I), and aconcomitant increase in myofiber sizes (FIGS. 4J-4K) fourteen dayspost-injury after a single dmPGE2 injection. Thus, exposure solely todmPGE2 impacts the magnitude and time course of the endogenous repair.

As the ultimate test, we determined if dmPGE2 enhanced regenerationcould lead to increased muscle strength after a natural injury inducedby downhill treadmill-running. In this scenario, damage was caused by adaily 10 min run on a downhill treadmill 20 degree decline²⁵. Duringweek one, aged mice in the treatment group ran for 5 days in successionand were injected daily with dmPGE2 after exercise. During week two,aged mice in the treatment group ran for 5 consecutive days but receivedno additional treatment (FIG. 4L). The specific twitch and tetanic forcewere compared for dmPGE2 treated and untreated gastrocnemius mousemuscles (GA) and both were significantly increased (FIGS. 4M-4P). Thus,an acute exposure to dmPGE2 concurrent with exercise-induced injury canconfer a significant increase in aged muscle strength.

We have discovered a new indication for PGE2 in skeletal muscleregeneration. Prior studies of PGE2 effects on skeletal muscle haveshown that it alters the proliferation, fusion, protein degradation, anddifferentiation of myoblasts in tissue culture²⁶⁻³⁰. Thus, these studiesdiffer from ours as myoblasts are progenitors that have lost stem cellfunction. Satellite cells (MuSCs) are crucial to development andregeneration^(3-8,31) and their numbers are increased by running orother high intensity exercise in young and aged mice andhumans^(15,32-36). Non-steroidal anti-inflammatory agents have beenreported to attenuate the exercise-induced increase in MuSCs^(15,32-36).Our data provide novel evidence that the beneficial effects of the earlytransient wave of inflammation that characterizes efficacious muscleregeneration¹⁵ is due in part to PGE2 and its receptor EP4, which areessential and sufficient for MuSC proliferation and engraftment. Forhematopoietic, liver, and colon tissues, delivery of the inhibitor of15-PGDH, SW033291, was recently shown to enhance regeneration²².Notably, PGE2 and its analogues have safely been used in human patientsfor decades, for instance to induce labor³⁷ and to promote hematopoieticstem cell transplantation³⁸ paving the way for its clinical use inrestoring muscles post-injury. In summary, our findings show that anacute PGE2 regimen suffices to rapidly and robustly enhance regenerationof exercise-induced damage and overcome age-associated limitationsleading to increased strength.

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Methods Mice

We performed all experiments and protocols in compliance with theinstitutional guidelines of Stanford University and Administrative Panelon Laboratory Animal Care (APLAC). We obtained wild-type aged C57BL/6(18-20 mo) mice from the US National Institute on Aging (NIA) for agedmuscle studies and young wild-type C57BL/6 mice from Jackson Laboratory.Double-transgenic GFP/luc mice were generated as described previously¹.Briefly, mice expressing a firefly luciferase (luc) transgene under theregulation of the ubiquitous Actb promoter were maintained in the FVBstrain. Mice expressing a green fluorescent protein (GFP) transgeneunder the regulation of the ubiquitous UBC promoter were maintained inthe C57BL/6 strain. We used cells from GFP/luc for allogenictransplantation experiments into NOD-SCID (Jackson Laboratory) recipientmice. EP4^(flox/flox) (EP4^(f/f)) mice were a kind gift from K.Andreasson (Stanford University)². Double-transgenicPax7^(CreERT2);Rosa26-LSL-Luc were generated by crossing Pax7^(CreERT2)mice obtained from Jackson Laboratory (Stock #017763)³ andRosa26-LSL-Luc obtained from Jackson Laboratory (Stock #005125)⁴. Wevalidated these genotypes by appropriate PCR-based strategies. All micefrom transgenic strains were of young age. Young mice were 2-4 mo. ofage and aged mice were 18-20 mo of age for all strains. All mice used inthese studies were females.

Muscle Stem Cell Isolation

We isolated and enriched muscle stem cells as previouslydescribed^(1,5,6). Briefly, a gentle collagenase digestion and mincingby the MACs Dissociator enabled numerous single fibers to bedissociated, followed by dispase digestion to release mononucleatedcells from their niches. Subsequently, the cell mixture was depleted forhematopoietic lineage expressing and non-muscle cells(CD45⁻/CD11b⁻/CD31⁻) using a magnetic bead column (Miltenyi). Theremaining cell mixture was then subjected to FACS analysis to sort forMuSCs co-expressing CD34 and α7-integrin markers. We generated andanalyzed flow cytometry scatter plots using FlowJo v10.0. For each sort,we pooled together MuSCs (˜5,000 each) from at least three independentdonor female mice.

Muscle Stem Cell Transplantation

We transplanted 250 MuSCs (FIGS. 3a, 3c and 3d ) or 1,000 MuSCs (FIG. 3b) immediately following FACS isolation or after collection from cellculture directly into the tibialis anterior (TA) muscles of recipientmice as previously described^(1,5,6). For young MuSC studies, wetransplanted cells from GFP/luc mice (2-4 mo of age) intohindlimb-irradiated NOD-SCID mice. For aged MuSCs studies, wetransplanted cells from aged C57BL/6 mice (18-20 mo, NIH) that weretransduced with a luc-IRES-GFP lentivirus (GFP/luc virus) on day 2 ofculture for a period of 24 hr before transplantation, as previouslydescribed⁵ (see below “Muscle stem cell culture, treatment andlentiviral infection” section for details). Prior to transplantation ofmuscle stem cells, we anesthetized NOD-SCID recipient mice with ketamine(2.4 mg per mouse) by intraperitoneal injection. We then irradiatedhindlimbs with a single 18 Gy dose, with the rest of the body shieldedin a lead jig. We performed transplantations within 2 d of irradiation.

Cultured cells were treated as indicated (vehicle or PGE2 treated 10ng/ml) and collected from hydrogel cultures by incubation with 0.5%trypsin in PBS for 2 min at 37° C. and counted using a hemocytometer. Weresuspended cells at desired cell concentrations in 0.1% gelatin/PBS andthen transplanted them (250 MuSCs per TA) by intramuscular injectioninto the TA muscles in a 10 μl volume. For fresh MuSCs transplantation,we coinjected sorted cells with 13 nmol of 16,16-Dimethyl ProstaglandinE2 (dmPGE2) (Tocris, catalog #4027) or vehicle control (PBS). Wecompared cells from different conditions by transplantation into the TAmuscles of contralateral legs in the same mice. One month aftertransplant, we injected 10 μl of notexin (10 μg ml⁻¹; Latoxan, France)to injure recipient muscles and to activate MuSCs in vivo. Eight weeksafter transplantation, mice were euthanized and the TAs were collectedfor analysis.

Bioluminescence Imaging

We performed bioluminescence imaging (BLI) using a Xenogen-100 system,as previously described^(1,5,6). Briefly, we anesthetized mice usingisofluorane inhalation and administered 120 μL D-luciferin (0.1 mmolkg⁻¹, reconstituted in PBS; Caliper LifeSciences) by intraperitonealinjection. We acquired BLI using a 60 s exposure at F-stop=1.0 at 5minutes after luciferin injection. Digital images were recorded andanalyzed using Living Image software (Caliper LifeSciences). We analyzedimages with a consistent region-of-interest (ROI) placed over eachhindlimb to calculate a bioluminescence signal. We calculated abioluminescence signal in radiance (p s⁻¹ cm⁻² sr⁻¹) value of 10⁴ todefine an engraftment threshold. This radiance threshold of 10⁴ isapproximately equivalent to the total flux threshold in p/s reportedpreviously. This BLI threshold corresponds to the histological detectionof one or more GFP+ myofibers^(1,5,6). We performed BLI imaging everyweek after transplantation.

Muscle Injury

We used an injury model entailing intramuscular injection of 10 μl ofnotexin (10 μg ml⁻¹; Latoxan) or cardiotoxin (10 μM; Latoxan) into theTA muscle. For cryoinjury, an incision was made in the skin overlyingthe TA muscle and a copper probe, chilled in liquid nitrogen, wasapplied to the TA muscle for three 10 s intervals, allowing the muscleto thaw between each application of the cryoprobe. When indicated, 48 hrafter injury either 16,16-Dimethyl Prostaglandin E2 (dmPGE2) (13 nmol,Tocris, catalog #4027) or vehicle control (PBS) was injected into the TAmuscle. The contralateral TA was used as an internal control. Wecollected tissues 14 days post-injury for analysis.

For Pax7^(CreERT2); Rosa26-LSL-Luc mice experiments, we treated micewith five consecutive daily intraperitoneal injections of tamoxifen toactivate luciferase expression under the control of the Pax7 promoter. Aweek after the last tamoxifen injection, mice were subjected tointramuscular injection of 10 μl of cardiotoxin (10 μM; Latoxan), whichwe designated as day 0 of the assay. Three days later either 13 nmoldmPGE2 (13 nmol) or vehicle control (PBS) was injected into the TAmuscle. The contralateral TA was used as an internal control.Bioluminescence was assayed at days 3, 7, 10 and 14 post-injury.

Tissue Histology

We collected and prepared recipient TA muscle tissues for histology aspreviously described^(5,6). We incubated transverse sections withanti-LAMININ (Millipore, clone A5, catalog #05-206, 1:200), andanti-PAX7 (Santa Cruz Biotechnology, catalog #sc-81648, 1:50) primaryantibodies and then with AlexaFluor secondary Antibodies (JacksonImmunoResearch Laboratories, 1:200). We counterstained nuclei with DAPI(Invitrogen). We acquired images with an AxioPlan2 epifluorescentmicroscope (Carl Zeiss Microimaging) with Plan NeoFluar 10×/0.30NA or20×/0.75NA objectives (Carl Zeiss) and an ORCA-ER digital camera(Hamamatsu Photonics) controlled by the SlideBook (3i) software. Theimages were cropped using Adobe Photoshop with consistent contrastadjustments across all images from the same experiment. The imagecomposites were generated using Adobe Illustrator. We analyzed thenumber of PAX7 positive cells using the MetaMorph Image Analysissoftware (Molecular Devices), and the fiber area using the BaxterAlgorithms for Myofiber Analysis that identified the fibers andsegmented the fibers in the image to analyze the area of each fiber. ForPAX7 quantification we examined serial sections spanning a depth of atleast 2 mm of the TA. For fiber area at least 10 fields ofLAMININ-stained myofiber cross-sections encompassing over 400 myofiberswere captured for each mouse as above. Data analyses were blinded. Theresearchers performing the imaging acquisition and scoring were unawareof treatment condition given to sample groups analyzed.

Hydrogel Fabrication

We fabricated polyethylene glycol (PEG) hydrogels from PEG precursors,synthesized as described previously⁶. Briefly, we produced hydrogels byusing the published formulation to achieve 12-kPa (Young's modulus)stiffness hydrogels in 1 mm thickness which is the optimal condition forculturing MuSCs and maintaining stem cell fate in culture⁶. Wefabricated hydrogel microwell arrays of 12-kPa for clonal proliferationexperiments, as described previously⁶. We cut and adhered all hydrogelsto cover the surface area of 12-well or 24-well culture plates.

Muscle Stem Cell Culture, Treatment and Lentiviral Infection

Following isolation, we resuspended MuSCs in myogenic cell culturemedium containing DMEM/F10 (50:50), 15% FBS, 2.5 ng ml⁻¹ fibroblastgrowth factor-2 (FGF-2 also known as bFGF) and 1%penicillin-streptomycin. We seeded MuSC suspensions at a density of 500cells per cm² surface area. We maintained cell cultures at 37° C. in 5%CO₂ and changed medium daily. For PGE2, 15-PGDH inhibitor and EP4receptor antagonist treatment studies, we added 1-200 ng/mlProstaglandin E2 (Cayman Chemical) (unless specified in the figurelegends, 10 ng/ml was the standard concentration used), and/or 1 μM EP4antagonist (ONO-AE3-208, Cayman Chemical), or 1 μM 15-PGDH inhibitor(SW033291, Cayman Chemical) to the MuSCs cultured on collagen coateddishes for the first 24 h. The cells were then trypsinized and cellsreseeded onto hydrogels for an additional 6 days of culture. Alltreatments were compared to their solvent (DMSO) vehicle control. Forstripped serum assays, we resuspended isolated MuSCs in mediumcontaining DMEM/F10 (50:50), 15% charcoal stripped FBS (Gibco, cat#12676011), 2.5 ng ml⁻¹ bFGF and 1% penicillin-streptomycin. When notedin the figure, we additionally added 1.5 μg/ml insulin (Sigma, 10516)and 0.25 μM dexamethasone (Sigma, D8893) to stripped serum cell medium.For these experiments MuSCs were cultured on hydrogels and vehicle(DMSO) or 10 ng/ml PGE2 (Cayman Chemical) was added to the cultures withevery media change (every two days). Proliferation (see below) wasassayed 7 days later.

We performed all MuSC culture assays and transplantations after 1 weekof culture unless noted otherwise. For aged MuSCs transplant studies, weinfected MuSCs with lentivirus encoding elongation factor-1αpromoter-driven luc-IRES-GFP (GFP/luc virus) for 24 h in culture asdescribed previously³. For EP4^(f/f) MuSCs studies, we isolated MuSCs asdescribed above (Muscle stem cell isolation), and infected all cellswith the GFP/luc virus and a subset of them was coinfected with alentivirus encoding pLM-CMV-R-Cre (mCherry/Cre virus) for 24 h inculture. pLM-CMV-R-Cre was a gift from Michel Sadelain (Addgene plasmid#27546)⁷. We transplanted aged MuSC (250 cells) or EP4^(f/f) MuSCs(1,000 cells) into young (2-4 mo) 18-gy irradiated TAs of NOD-SCIDrecipient mice. For in vitro proliferation assays, EP4^(f/f) MuSCs wereplated on hydrogels post-infection and treated for 24 hr with vehicle(DMSO) or 10 ng/ml PGE2, and proliferation was assayed 3 days later.Cells were assayed for GFP and/or mCherry expression 48 h post-infectionusing an inverted fluorescence microscope (Carl Zeiss Microimaging).MuSCs are freshly isolated from the mice by FACS and put in culture fora maximum time period of one week, therefore mycoplasma contamination isnot assessed.

Proliferation Assays

To assay proliferation, we used three different assays (hemocytometer,VisionBlue, and EdU). For each, we seeded MuSCs on flat hydrogels(hemocytometer and VisionBlue) or collagen-coated plates (EdU assay) ata density of 500 cells per cm² surface area. For hemocytometer cellnumber count, we collected cells at indicated timepoints by incubationwith 0.5% trypsin in PBS for 5 min at 37° C. and quantified them using ahemocytometer at least 3 times. Additionally, we used the VisionBlueQuick Cell Viability Fluorometric Assay Kit (BioVision, catalog #K303)as a readout for cell growth in culture. Briefly, we incubated MuSCswith 10% VisionBlue in culture medium for 3 h, and measured fluorescenceintensity on a fluorescence plate reader (Infinite M1000 PRO, Tecan) atEx=530-570 nm, Em=590-620 nm. We assayed proliferation using theClick-iT EdU Alexa Fluor 555 Imaging kit (Life Technologies). Briefly,we incubated live cells with EdU (20 μM) for 1 hr prior to fixation, andstained nuclei according to the manufacturer's guidelines together withanti-MYOGENIN (Santa Cruz, catalog #sc576, 1:250) to assaydifferentiation. We counterstained nuclei with DAPI (Invitrogen). Weacquired images with an AxioPlan2 epifluorescent microscope (Carl ZeissMicroimaging) with Plan NeoFluar 10×/0.30NA or 20×/0.75NA objectives(Carl Zeiss) and an ORCA-ER digital camera (Hamamatsu Photonics)controlled by SlideBook (3i) software. We quantified EdU positive cellsusing the MetaMorph Image Analysis software (Molecular Devices). Dataanalyses were blinded, where researchers performing cell scoring wereunaware of the treatment condition given to sample groups analyzed.

Clonal Muscle Stem Cell Proliferation and Fate Analyses

We assayed clonal muscle stem cell proliferation by time-lapsemicroscopy as previously described^(5,6). Briefly, we treated isolatedaged MuSCs with PGE2 (Cayman Chemical) or vehicle (DMSO) for 24 hr.After five days of growth on hydrogels, cells were reseeded at a densityof 500 cells per cm² surface area in hydrogel microwells with 600 μmdiameter. For time-lapse microscopy we monitored cell proliferation forthose wells with single cells beginning 12 hr (day 0) to two days afterseeding and recorded images every 3 min at 10× magnification using aPALM/AxioObserver Z1 system (Carl Zeiss MicroImaging) with a customenvironmental control chamber and motorized stage. We changed mediumevery other day in between the acquisition time intervals. We analyzedtime-lapse image sequences using the Baxter Algorithms for Cell Trackingand Lineage Reconstruction to identify and track single cells andgenerate lineage trees^(5,6,8-10).

Viable and dead cells were distinguished in time-lapse sequences basedon phase-contrast boundary and motility maintenance or loss,respectively. We found that the rates of proliferation (division) anddeath in the two conditions varied over time, Therefore, we estimatedthe rates for the first and the second 24 hour intervals separately. Thevalues were estimated using the equations described in⁶, and found inTable 1. We denote the proliferation rates in the two intervals p₂₄ andp₄₈ and the corresponding death rates d₂₄ and d₄₈. As an example, theproliferation rate in the treated condition during the second 24 hourinterval is 5.38% per hour. Table 1 (below) shows that the rates ofproliferation and death in the two conditions are similar in the firsttime interval, and that the difference in cell numbers at the end of theexperiment is due to differences in both the division rates and thedeath rates during the second time interval. The modeled cell counts inthe two time intervals are given by

${c(t)} = \left\{ \begin{matrix}{{c_{0}\mspace{14mu} {\exp \left( {\left( {p_{24} - d_{24}} \right)t} \right)}}\mspace{95mu}} & {\mspace{11mu} {0 \leq t \leq 24}} \\{{c(24)}\mspace{14mu} {\exp \left( {\left( {p_{48} - d_{48}} \right)\left( {t - 24} \right)} \right)}} & {24 < t \leq 48}\end{matrix} \right.$

where c₀ is the number of cells at the onset. The modeled curves areplotted together with the actual cell counts in FIG. 7F.

TABLE 1 Estimated proliferation and death rates per hours. p₂₄ p₄₈ d₂₄d₄₈ DMSO 0.0488 0.0403 0.0045 0.0112 E2 0.0475 0.0538 0.0067 0.0012

The data analysis was blinded. The researchers performing the imagingacquisition and scoring were unaware of the treatment condition given tosample groups analyzed.

Quantitative RT-PCR

We isolated RNA from MuSCs using the RNeasy Micro Kit (Qiagen). Formuscle samples, we snap froze the tissue in liquid nitrogen, homogenizedthe tissues using a mortar and pestle, followed by syringe and needletrituration, and then isolated RNA using Trizol (Invitrogen). Wereverse-transcribed cDNA from total mRNA from each sample using theSensiFAST™ cDNA Synthesis Kit (Bioline). We subjected cDNA to RT-PCRusing SYBR Green PCR Master Mix (Applied Biosystems) or TaqMan Assays(Applied Biosystems) in an ABI 7900HT Real-Time PCR System (AppliedBiosystems). We cycled samples at 95° C. for 10 min and then 40 cyclesat 95° C. for 15 s and 60° C. for 1 min. To quantify relative transcriptlevels, we used 2-ΔΔCt to compare treated and untreated samples andexpressed the results relative to Gapdh. For SYBR Green qRT-PCR, we usedthe following primer sequences: Gapdh. forward5′-TTCACCACCATGGAGAAGGC-3′ (SEQ ID NO: 1), reverse5′-CCCTTTTGGCTCCACCCT-3′ (SEQ ID NO: 2); Hpgd, forward5′-TCCAGTGTGATGTGGCTGAC-3′ (SEQ ID NO: 3), reverse5′-ATTGITCACGCCTGCATTGT-3′ (SEQ ID NO: 4); Ptges, forward5′-GCTGTCATCACAGGCCAGA-3′ (SEQ ID NO: 5), reverse5′-CTCCACATCTGGGTCACTCC-3′ (SEQ ID NO: 6); Ptges2, forward5′-CTCCTACAGGAAAGTGCCCA-3′ (SEQ ID NO: 7), reverse5′-ACCAGGTAGGTCITGAGGGC-3′ (SEQ ID NO: 8); Ptger1, forward 5′GTGGTGTCGTGCATCTGCT-3′ (SEQ ID NO: 9), reverse, 5′CCGCTGCAGGGAGTTAGAGT-3′ (SEQ ID NO: 10), and Ptger2, forward5′-ACCTTCGCCATATGCTCCIT-3′ (SEQ ID NO: 11), reverse5′-GGACCGGTGGCCTAAGTATG-3′ (SEQ ID NO: 12). TaqMan Assays (AppliedBiosystems) were used to quantify Pax7. Myogenin. Slco2a1 (PGT), Ptger3and Ptger4 in samples according to the manufacturer instructions withthe TaqMan Universal PCR Master Mix reagent kit (Applied Biosystems).Transcript levels were expressed relative to Gapdh levels. For SYBRGreen qPCR, Gapdh qPCR was used to normalize input cDNA samples. ForTaqman qPCR, multiplex qPCR enabled target signals (FAM) to benormalized individually by their internal Gapdh signals (VIC).

PGE2 Elisa

Muscle was harvested, rinsed in ice-cold PBS containing indomethacin(5.6 μg/ml), and snap frozen in liquid nitrogen. Frozen samples werepulverized in liquid nitrogen. The powder was transferred to anEppendorf tube with 500 μl of lysate buffer (50 mM Tris-HCl pH 7.5, 150mM NaCl, 4 mM CaCl, 1.5% Triton X-100, protease inhibitors andmicrococcal nuclease), and then homogenized using a tissue homogenizer.The PGE2 level of the supernatant was measured using a PGE2 ELISA Kit(R&D Systems, catalog #KGE004B) and expressed relative to total proteinmeasured by BCA assay (BioRad) and expressed as ng of PGE2. Each samplewas assayed in duplicate and in each of two independent experiments.

cAMP Activity Assay

MuSCs were treated with DMSO (vehicle) or PGE2 (10 ng/ml) for 1 h andcyclic AMP levels measured according to the cAMP-Glo Assay protocoloptimized by the manufacturer (Promega). Each sample was assayed intriplicate and in two independent experiments.

Flow Cytometry

We assayed Annexin V as a readout of apoptosis for MuSCs after 7 days inculture on hydrogels, after an initial acute (24 hr) treatment ofvehicle (DMSO) or PGE2 (10 ng/ml). We used the FITC Annexin V ApoptosisDetection Kit (Biolegend, cat #640914) according to the protocol of themanufacturer. We analyzed the cells for Annexin V on a FACS LSR IIcytometer using FACSDiva software (BD Biosciences) in the Shared FACSFacility, purchased using an NIH S10 Shared Instrument Grant(S10RR027431-01).

Mass Spectrometry

Analytes:

All prostaglandin standards—PGF2α; PGE2; PGD2; 15-keto PGE2;13,14-dihydro 15-keto PGE2; PGE2-D4; and PGF2α-D9—were purchased fromCayman Chemical. For the PGE2-D4 internal standard, positions 3 and 4were labeled with a total of four deuterium atoms. For PGF2α-D9,positions 17, 18, 19 and 20 were labeled with a total of nine deuteriumatoms.

Calibration Curve Preparation:

Analyte stock solutions (5 mg/mL) were prepared in DMSO. These stocksolutions were serially diluted with acetonitrile/water (1:1 v/v) toobtain a series of standard working solutions, which were used togenerate the calibration curve. Calibration curves were prepared byspiking 10 uL of each standard working solution into 200 μL ofhomogenization buffer (acetone/water 1:1 v/v; 0.005% BHT to preventoxidation) followed by addition of 10 uL internal standard solution(3000 ng/mL each PGF2α-D9 and PGE2-D4). A calibration curve was preparedfresh with each set of samples. Calibration curve ranges: for PGE2 and13,14-dihydro 15-keto PGE2, from 0.05 ng/mL to 500 ng/mL; for PGD2 andPGF2α, from 0.1 ng/mL to 500 ng/mL; and for 15-keto PGE2, from 0.025ng/mL to 500 ng/mL.

Extraction Procedure:

The extraction procedure was modified from that of Prasain et al.¹¹ andincluded acetone protein precipitation followed by 2-step liquid-liquidextraction; the latter step enhances LC-MS/MS sensitivity. Butylatedhydroxytoluene (BHT) and evaporation under nitrogen (N2) gas were usedto prevent oxidation.

Solid tissues were harvested, weighed, and snap-frozen with liquidnitrogen. Muscle tissue was combined with homogenization beads and 200μL homogenization buffer in a polypropylene tube and processed in aFastPrep 24 homogenizer (MP Biomedicals) for 40 seconds at a speed of 6m/s. After homogenization, 10 μL internal standard solution (3000 ng/mL)was added to tissue homogenate followed by sonication and shaking for 10minutes. Samples were centrifuged and the supernatant was transferred toa clean eppendorf tube. 200 μL hexane was added to the sample, followedby shaking for 15 minutes, then centrifugation. Samples were frozen at−80° C. for 40 minutes. The hexane layer was poured off from the frozenlower aqueous layer, and discarded. After thawing, 25 μL of IN formicacid was added to the bottom aqueous layer, and the samples werevortexed. For the second extraction, 200 μL chloroform was added to theaqueous phase. Samples were shaken for 15 minutes to ensure fullextraction. Centrifugation was performed to separate the layers. Thelower chloroform layer was transferred to a new eppendorf tube andevaporated to dryness under nitrogen at 40° C. The dry residue wasreconstituted in 100 μL acetonitrile/10 mM ammonium acetate (2:8 v/v)and analyzed by LC-MS/MS.

LC-MS/MS:

Since many prostaglandins are positional isomers with identical massesand have similar fragmentation patterns, chromatographic separation iscritical. Two SRM transitions—one quantifier and one qualifier—werecarefully selected for each analyte. Distinctive qualifier ion intensityratios and retention times were essential to authenticate the targetanalytes. All analyses were carried out by negative electrosprayLC-MS/MS using an LC-20ADxa prominence liquid chromatograph and 8030triple quadrupole mass spectrometer (Shimadzu). HPLC conditions: AcquityUPLC BEH C18 2.1×100 mm, 1.7 um particle size column was operated at 50°C. with a flow rate of 0.25 mL/min. Mobile phases consisted of A: 0.1%acetic acid in water and B: 0.1% acetic acid in acetonitrile. Elutionprofile: initial hold at 35% B for 5 minutes, followed by a gradient of35%-40% in 3 minutes, then 40%-95% in 3 minutes; total run time was 14minutes. Injection volume was 20 uL. Using these HPLC conditions, weachieved baseline separation of the analytes of interest.

Selected reaction monitoring (SRM) was used for quantification. The masstransitions were as follows: PGD2: m/z 351.10→m/z 315.15 (quantifier)and m/z 351.10→m/z 233.05 (qualifier); PGE2: m/z 351.10→m/z 271.25(quantifier) and m/z 351.10→m/z 315.20 (qualifier); PGF2α: m/z353.10→m/z 309.20 (quantifier) and m/z 353.10→m/z 193.20 (qualifier); 15keto-PGE2: m/z 349.30→m/z 331.20 (quantifier) and m/z 349.30→m/z 113.00(qualifier); 13,14-dihydro 15-keto PGE2: m/z 351.20→m/z 333.30(quantifier) and m/z 351.20→m/z 113.05 (qualifier); PGE2-D4: m/z355.40→m/z 275.20; and PGF2α-D9: m/z 362.20→m/z 318.30. Dwell time was20-30 ms.

Quantitative analysis was done using LabSolutions LCMS (Shimadzu). Aninternal standard method was used for quantification: PGE2-D4 was usedas an internal standard for quantification of PGE2, 15-keto PGE2, and13,14-dihydro 15-keto PGE2. PGF2α-D9 was the internal standard forquantification of PGD2 and PGF2α. Calibration curves were linear(R>0.99) over the concentration range using a weighting factor of 1/X²where X is the concentration. The back-calculated standardconcentrations were ±15% from nominal values, and ±20% at the lowerlimit of quantitation (LLOQ).

In Vive Muscle Force Measurement

Aged mice (18 mo.) were subjected to downhill treadmill run for 2consecutive weeks. During week 1, mice ran daily for 5 days and restedon days 6 and 7. Two hours after each treadmill run during week 1, each(lateral and medial) gastrocnemius (GA) muscle from both legs of eachmouse was injected with a dose of either PBS (vehicle control) or 13 nMdmPGE2 (experimental group). During week 2, mice were subjected to 5days treadmill run only. The treadmill run was performed using theExer3/6 (Columbus Instruments). Mice ran for 10 minutes on the treadmillat 20 degrees downhill, starting at a speed of 7 meters/min. After 3min, the speed was increased by 1 meter/min to a final speed of 14meter/min. 10 minutes run time was chosen, as exhaustion defined as theinability of the animal to remain on the treadmill despite electricalprodding, was observed at a median of 12 minute in an independentcontrol aged mouse group. Force measurements were on the GA muscles atweek 5 based on a protocol published previously⁵. Briefly, for eachmouse, an incision was made to expose the GA. We severed the calcaneusbone with intact achilles tendon and attached the tendon-bone complex toa 300C-LR force transducer (Aurora Scientific) with a thin metal hook.The muscles and tendons were kept moist by periodic wetting with saline(0.9% sodium chloride) solution. The lower limb was immobilized belowthe knee by a metal clamp without compromising the blood supply to theleg. The mouse was under inhaled anesthetic (2% isofluorane) during theentire force measuring procedure and body temperature was maintained bya heat lamp. In all measurements, we used 0.1-ms pulses at apredetermined supramaximal stimulation voltage. The GA muscles werestimulated via the proximal sciatic nerve using a bipolar electricalstimulation cuff delivering a constant current of 2 mA (square pulsewidth 0.1 ms). GA muscles were stimulated with a single 0.1-ms pulse fortwitch force measurements, and a train of 150 Hz for 0.3 s pulses fortetanic force measurements. We performed five twitch and then fivetetanic measurements on each muscle, with 2-3 min recovery between eachmeasurement with n=5 mice per group. Data were collected with a PCI-6251acquisition card (National Instruments) and analyzed in Matlab. Wecalculated specific force values by normalizing the force measurementsby the muscle physiological cross-sectional areas (PCSAs), which weresimilar between the control and the experimental PGE2 treated group(Table 2). PCSA (measured in mm²) was calculated according to thefollowing equation¹²:

PCSA (mm²)=[mass (g)×Cos θ]÷[ρ(g/mm³)×fiber length (mm)],

where θ is pennation angle of the fiber and ρ is muscle density(0.001056 g/mm³).

Statistical Analyses

We performed cell culture experiments in at least three independentexperiments where three biological replicates were pooled in each. Ingeneral, we performed MuSC transplant experiments in at least twoindependent experiments, with at least 3-5 total transplants percondition. We used a paired t-test for experiments where control sampleswere from the same experiment in vitro or from contralateral limbmuscles in vivo. A non-parametric Mann-Whitney test was used todetermine the significance difference between untreated (−) vs treated(PGE or dmPGE2) groups using α=0.05. ANOVA or multiple t-test wasperformed for multiple comparisons with significance level determinedusing Bonferroni correction or with Fisher's test as indicated in thefigure legends. Unless otherwise described, data are shown as themean±s.e.m.

METHODS REFERENCES

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TABLE 2 Physiological cross-sectional area (PCSA) of aged gastrocnemiusweek 5 post-exercise. PCSA Pennation Fiber GA (medial + angle θ lengthMass lateral) Mouse ID Leg (degree) Cosine(θ) (mm) (g) (mm²) Control-1Left 21 0.93 6.88 0.18 23.13 Right 21 0.93 6.64 0.18 23.82 Control-2Left 26 0.90 4.03 0.16 33.79 Right 22 0.93 5.34 0.16 26.31 Control-3Left 21 0.93 4.52 0.15 29.34 Right 23 0.92 4.59 0.17 32.28 Control-4Left 24 0.91 5.07 0.14 23.89 Right 23 0.92 4.75 0.13 23.86 Control-5Left 19 0.95 6.07 0.16 17.75 Right 18 0.95 6.05 0.15 10.25 dmPGE2-1 Left12 0.98 7.60 0.25 30.47 Right Tendon — — — — damage dmPGE2-2 Left 120.96 4.85 0.16 30.56 Right 16 0.91 4.80 0.14 26.55 dmPGE2-3 Left 14 0.975.89 0.17 26.52 Right 13 0.94 5.63 0.14 22.94 dmPGE2-4 Left 14 0.97 6.670.14 19.29 Right 13 0.97 7.74 0.16 19.07 dmPGE2-5 Left 11 0.98 5.56 0.1728.42 Right 11 0.98 5.54 0.16 26.85 Avg. Control 25.09 Avg. dmPGE2 25.63

Example 2: Increased Muscle Forces after Prostaglandin E2 (PGE2)Injection

This example shows an increase in specific twitch force of gastrocnemiusmuscles in aged mice injected with PGE2. The aged mice (18 months old)were subject to treadmill run to exhaustion daily for 10 days. Thetreadmill run was performed using the Exer3/6 (Columbus Instruments).Mice ran on the treadmill at 20 degrees downhill, starting at a speed of10 meters/min. After 3 min, the speed was increased 1 meter/min to afinal speed of 20 meters/min. Exhaustion was defined as the inability ofthe animal to remain on the treadmill despite electrical prodding. 2 hafter each treadmill run, both gastrocnemius muscles of each mouse wereinjected with either PBS (control group) or 3 nM PGE2 (experimentalgroup). The force measurement was performed 4 weeks after the lasttreadmill run using a 300C-LR force transducer (Aurora Scientific) witha single 0.1 ms pulse at predetermined supramaximal stimulationintensity.

Representative raw muscle force traces of single gastrocnemius musclesare provided in FIGS. 4M-4N. The muscle force and synchronization pulseswere recorded via a PCI-6251 acquisition card (National Instruments) andanalyzed using Matlab. FIGS. 4O-4P show the specific muscle force valuesthat were calculated by normalizing the force measurements with themuscle physiological cross-sectional area. The specific twitch forcevalues (kN/m²) are represented by the Box and Whiskers plot that showsthe minimum, maximum, and median values. Five repetitive measurementswere made from each muscle. N=4 for the control group and n=5 for thePGE2 injected group. ** represents a statistical significant value ofp<0.005 by 2-tailed Mann Whitney test.

VI. Exemplary Embodiments

Exemplary embodiments provided in accordance with the presentlydisclosed subject matter include, but are not limited to, the claims andthe following embodiments:

1. A method for stimulating the proliferation of a population ofisolated muscle cells, the method comprising:

-   -   culturing the population of isolated muscle cells with a        compound selected from the group consisting of prostaglandin E2        (PGE2), a PGE2 prodrug, a PGE2 receptor agonist, a compound that        attenuates PGE2 catabolism, a compound that neutralizes PGE2        inhibition, a derivative thereof, an analog thereof, and a        combination thereof.        2. The method of embodiment 1, wherein the population of        isolated muscle cells is purified.        3. The method of embodiment 1 or 2, wherein the population of        isolated muscle cells comprises skeletal muscle cells, smooth        muscle cells, cardiac muscle cells, embryonic stem cell-derived        muscle cells, induced pluripotent stem cell-derived muscle        cells, dedifferentiated muscle cells, or a combination thereof.        4. The method of any one of embodiments 1 to 3, wherein the        population of isolated muscle cells comprises muscle stem cells,        satellite cells, myocytes, myoblasts, myotubes, myofibers, or a        combination thereof.        5. The method of any one of embodiments 1 to 4, wherein the        population of isolated muscle cells is obtained from a subject.        6. The method of embodiment 5, wherein the subject has a        condition or disease associated with muscle damage, injury, or        atrophy.        7. The method of embodiment 6, wherein the condition or disease        associated with muscle damage, injury, or atrophy is selected        from the group consisting of acute muscle injury or trauma, soft        tissue hand injury, Duchenne muscular dystrophy (DMD), Becker        muscular dystrophy, limb girdle muscular dystrophy, amyotrophic        lateral sclerosis (ALS), distal muscular dystrophy (DD),        inherited myopathies, myotonic muscular dystrophy (MDD),        mitochondrial myopathies, myotubular myopathy (MM), myasthenia        gravis (MG), congestive heart failure, periodic paralysis,        polymyositis, rhabdomyolysis, dermatomyositis, cancer cachexia,        AIDS cachexia, cardiac cachexia, stress induced urinary        incontinence, and sarcopenia.        8. The method of any one of embodiments 1 to 7, wherein the PGE2        derivative comprises 16,16-dimethyl prostaglandin E2.        9. The method of any one of embodiments 1 to 7, wherein the        compound that attenuates PGE2 catabolism comprises a compound,        neutralizing peptide, or neutralizing antibody that inactivates        or blocks 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or        inactivates or blocks a prostaglandin transporter (PTG or        SLCO2A1).        10. The method of any one of embodiments 1 to 9, wherein        culturing the population of isolated muscle cells with the        compound comprises acute, intermittent, or continuous exposure        of the population of isolated muscle cells to the compound.        11. A method for promoting muscle cell engraftment in a subject,        the method comprising:    -   culturing a population of isolated muscle cells with an        effective amount of a compound selected from the group        consisting of prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2        receptor agonist, a compound that attenuates PGE2 catabolism, a        compound that neutralizes PGE2 inhibition, a derivative thereof,        an analog thereof, and a combination thereof, to promote        engraftment of the muscle cells in the subject; and    -   administering the cultured muscle cells to the subject.        12. The method of embodiment 11, wherein the population of        isolated muscle cells is purified.        13. The method of embodiment 11 or 12, wherein the population of        isolated muscle cells comprises skeletal muscle cells, smooth        muscle cells, cardiac muscle cells, embryonic stem cell-derived        muscle cells, induced pluripotent stem cell-derived muscle        cells, dedifferentiated muscle cells, or a combination thereof.        14. The method of any one of embodiments 11 to 13, wherein the        population of isolated muscle cells comprises muscle stem cells,        satellite cells, myocytes, myoblasts, myotubes, myofibers, or a        combination thereof.        15. The method of any one of embodiments 11 to 14, wherein the        population of isolated muscle cells is autologous to the        subject.        16. The method of any one of embodiments 11 to 14, wherein the        population of isolated muscle cells is allogeneic to the        subject.        17. The method of any one of embodiments 11 to 16, wherein the        subject has a condition or disease associated with muscle        damage, injury, or atrophy.        18. The method of embodiment 17, wherein the condition or        disease associated with muscle damage, injury, or atrophy is        selected from the group consisting of acute muscle injury or        trauma, soft tissue hand injury, Duchenne muscular dystrophy        (DMD), Becker muscular dystrophy, limb girdle muscular        dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular        dystrophy (DD), inherited myopathies, myotonic muscular        dystrophy (MDD), mitochondrial myopathies, myotubular myopathy        (MM), myasthenia gravis (MG), congestive heart failure, periodic        paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer        cachexia, AIDS cachexia, cardiac cachexia, stress induced        urinary incontinence, and sarcopenia.        19. The method of any one of embodiments 11 to 18, wherein the        PGE2 derivative comprises 16,16-dimethyl prostaglandin E2.        20. The method of any one of embodiments 11 to 18, wherein the        compound that attenuates PGE2 catabolism comprises a compound,        neutralizing peptide, or neutralizing antibody that inactivates        or blocks 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or        inactivates or blocks a prostaglandin transporter (PTG or        SLCO2A1).        21. The method of any one of embodiments 11 to 20, wherein        culturing the population of isolated muscle cells with the        compound comprises acute, intermittent, or continuous exposure        of the population of isolated muscle cells to the compound.        22. A composition comprising a population of isolated muscle        cells and a compound selected from the group consisting of        prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor        agonist, a compound that attenuates PGE2 catabolism, a compound        that neutralizes PGE2 inhibition, a derivative thereof, an        analog thereof, and a combination thereof.        23. The composition of embodiment 22, wherein the population of        isolated muscle cells comprises skeletal muscle cells, smooth        muscle cells, cardiac muscle cells, embryonic stem cell-derived        muscle cells, induced pluripotent stem cell-derived muscle        cells, dedifferentiated muscle cells, or a combination thereof.        24. The composition of embodiment 22 or 23, wherein the        population of isolated muscle cells comprises muscle stem cells,        satellite cells, myocytes, myoblasts, myotubes, myofibers, or a        combination thereof.        25. The composition of any one of embodiments 22 to 24, further        comprising a pharmaceutically acceptable carrier.        26. A kit comprising the composition of any one of embodiments        22 to 25, and an instruction manual.        27. A method for regenerating a population of muscle cells in a        subject having a condition or disease associated with muscle        damage, injury, or atrophy, the method comprising:    -   administering to the subject a therapeutically effective amount        of a compound selected from the group consisting of        prostaglandin E2 (PGE2), a PGE2 prodrug, a PGE2 receptor        agonist, a compound that attenuates PGE2 catabolism, a compound        that neutralizes PGE2 inhibition, a derivative thereof, an        analog thereof, and a combination thereof, and a        pharmaceutically acceptable carrier, to increase the population        of muscle cells and/or to enhance muscle function in the        subject.        28. The method of embodiment 27, wherein the condition or        disease associated with muscle damage, injury, or atrophy is        selected from the group consisting of acute muscle injury or        trauma, soft tissue hand injury, Duchenne muscular dystrophy        (DMD), Becker muscular dystrophy, limb girdle muscular        dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular        dystrophy (DD), inherited myopathies, myotonic muscular        dystrophy (MDD), mitochondrial myopathies, myotubular myopathy        (MM), myasthenia gravis (MG), congestive heart failure, periodic        paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer        cachexia, AIDS cachexia, cardiac cachexia, stress induced        urinary incontinence, and sarcopenia.        29. The method of embodiment 27 or 28, wherein the population of        muscle cells comprises skeletal muscle cells, smooth muscle        cells, cardiac muscle cells, embryonic stem cell-derived muscle        cells, induced pluripotent stem cell-derived muscle cells,        dedifferentiated muscle cells, or a combination thereof.        30. The method of any one of embodiments 27 to 29, wherein the        population of muscle cells comprises muscle stem cells,        satellite cells, myocytes, myoblasts, myotubes, myofibers, or a        combination thereof.        31. The method of any one of embodiments 27 to 30, wherein the        PGE2 derivative comprises 16,16-dimethyl prostaglandin E2.        32. The method of any one of embodiments 27 to 30, wherein the        compound that attenuates PGE2 catabolism comprises a compound,        neutralizing peptide, or neutralizing antibody that inactivates        or blocks 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or        inactivates or blocks a prostaglandin transporter (PTG or        SLCO2A1).        33. The method of any one of embodiments 27 to 32, wherein        administering the compound comprises oral, intraperitoneal,        intramuscular, intra-arterial, intradermal, subcutaneous,        intravenous, or intracardiac administration.        34. The method of any one of embodiments 27 to 33, wherein the        compound is administered in accordance with an acute regimen.        35. The method of any one of embodiments 27 to 34, wherein        administering further comprises administering a population of        isolated muscle cells to the subject.        36. The method of embodiment 35, wherein the population of        isolated muscle cells is autologous to the subject.        37. The method of embodiment 35, wherein the population of        isolated muscle cells is allogeneic to the subject.        38. The method of any one of embodiments 35 to 37, wherein the        population of isolated muscle cells is purified.        39. The method of any one of embodiments 35 to 38, wherein the        population of isolated muscle cells is cultured with the        compound prior to being administered to the subject.        40. The method of embodiment 39, wherein culturing the        population of isolated muscle cells with the compound comprises        acute, intermittent, or continuous exposure of the population of        isolated muscle cells to the compound.        41. The method of any one of embodiments 35 to 40, wherein        administering the population of isolated muscle cells comprises        injecting or transplanting the cells into the subject.        42. The method of any one of embodiments 35 to 41, wherein the        population of isolated muscle cells and the compound are        administered to the subject concomitantly.        43. The method of any one of embodiments 35 to 41, wherein the        population of isolated muscle cells and the compound are        administered to the subject sequentially.        44. A method for preventing or treating a condition or disease        associated with muscle damage, injury or atrophy in a subject in        need thereof, the method comprising: administering to the        subject (i) a therapeutically effective amount of a compound        selected from the group consisting of prostaglandin E2 (PGE2), a        PGE2 prodrug, a PGE2 receptor agonist, a compound that        attenuates PGE2 catabolism, a compound that neutralizes PGE2        inhibition, a derivative thereof, an analog thereof, and a        combination thereof, and a pharmaceutically acceptable carrier,        and (ii) a population of isolated muscle cells, to prevent or        treat the condition or disease associated with muscle damage,        injury, or atrophy.        45. The method of embodiment 44, wherein the PGE2 derivative        comprises 16,16-dimethyl prostaglandin E2.        46. The method of embodiment 44, wherein the compound that        attenuates PGE2 catabolism comprises a compound, neutralizing        peptide, or neutralizing antibody that inactivates or blocks        15-hydroxyprostaglandin dehydrogenase (15-PGDH) or inactivates        or blocks a prostaglandin transporter (PTG or SLCO2A1).        47. The method of any one of embodiments 44 to 46, wherein the        population of isolated muscle cells comprises skeletal muscle        cells, smooth muscle cells, cardiac muscle cells, embryonic stem        cell-derived muscle cells, induced pluripotent stem cell-derived        muscle cells, dedifferentiated muscle cells, or a combination        thereof.        48. The method of any one of embodiments 44 to 47, wherein the        population of isolated muscle cells comprises muscle stem cells,        satellite cells, myoblasts, myocytes, myotubes, myofibers, or a        combination thereof.        49. The method of any one of embodiments 44 to 48, wherein the        population of isolated muscle cells is purified.        50. The method of any one of embodiments 44 to 49, wherein the        population of isolated muscle cells is cultured with the        compound prior to being administered to the subject.        51. The method of embodiment 50, wherein culturing the        population of isolated muscle cells with the compound comprises        acute, intermittent, or continuous exposure of the population of        isolated muscle cells to the compound.        52. The method of any one of embodiments 44 to 51, wherein the        population of isolated muscle cells is autologous to the        subject.        53. The method of any one of embodiments 44 to 51, wherein the        population of isolated muscle cells is allogeneic to the        subject.        54. The method of any one of embodiments 44 to 53, wherein        administering the compound comprises oral, intraperitoneal,        intramuscular, intra-arterial, intradermal, subcutaneous,        intravenous, or intracardiac administration.        55. The method of any one of embodiments 44 to 54, wherein the        compound is administered in accordance with an acute regimen.        56. The method of any one of embodiments 44 to 55, wherein        administering the population of isolated muscle cells comprises        injecting or transplanting the cells into the subject.        57. The method of any one of embodiments 44 to 56, wherein the        compound and the population of isolated muscle cells are        administered to the subject concomitantly.        58. The method of any one of embodiments 44 to 56, wherein the        compound and the population of isolated muscle cells are        administered to the subject sequentially.        59. The method of any one of embodiments 44 to 58, wherein the        subject is suspected of having or at risk for developing the        condition or disease associated with muscle damage, injury, or        atrophy.        60. The method of any one of embodiments 44 to 59, wherein the        condition or disease associated with muscle damage, injury or        atrophy is selected from the group consisting of acute muscle        injury or trauma, soft tissue hand injury, Duchenne muscular        dystrophy (DMD), Becker muscular dystrophy, limb girdle muscular        dystrophy, amyotrophic lateral sclerosis (ALS), distal muscular        dystrophy (DD), inherited myopathies, myotonic muscular        dystrophy (MDD), mitochondrial myopathies, myotubular myopathy        (MM), myasthenia gravis (MG), congestive heart failure, periodic        paralysis, polymyositis, rhabdomyolysis, dermatomyositis, cancer        cachexia, AIDS cachexia, cardiac cachexia, stress induced        urinary incontinence, and sarcopenia.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

Informal Sequence Listing Gapdh, forward (SEQ ID NO: 1)5′-TTCACCACCATGGAGAAGGC-3′ Gapdh, reverse (SEQ ID NO: 2)5′-CCCTTTTGGCTCCACCCT-3′ Hpgd, forward (SEQ ID NO: 3)5′-TCCAGTGTGATGTGGCTGAC-3′  Hpgd, reverse (SEQ ID NO: 4)5′-ATTGTTCACGCCTGCATTGT-3′ Ptges, forward (SEQ ID NO: 5)5′-GCTGTCATCACAGGCCAGA-3′ Ptges, reverse (SEQ ID NO: 6)5′-CTCCACATCTGGGTCACTCC-3′ Ptges2, forward (SEQ ID NO: 7)5′-CTCCTACAGGAAAGTGCCCA-3′ Ptges2, reverse (SEQ ID NO: 8)5′-ACCAGGTAGGTCTTGAGGGC-3′ Ptger1, forward (SEQ ID NO: 9)5′ GTGGTGTCGTGCATCTGCT-3′ Ptger1, reverse (SEQ ID NO: 10)5′ CCGCTGCAGGGAGTTAGAGT-3′ Ptger2, forward (SEQ ID NO: 11)5′-ACCTTCGCCATATGCTCCTT-3′ Ptger2, reverse (SEQ ID NO: 12)5′-GGACCGGTGGCCTAAGTATG-3′

1-30. (canceled)
 31. A method of treating muscle damage, muscle injuryor muscle atrophy comprising administering a therapeutically effectiveamount of a composition comprising a hydroxyprostaglandin dehydrogenase(PGDH) inhibitor to a subject in need thereof, thereby treating saidmuscle damage, muscle injury or muscle atrophy.
 32. The method of claim31, wherein said PGDH inhibitor is a 15-hydroxyprostaglandindehydrogenase (15-PGDH) inhibitor.
 33. The method of claim 31, whereinsaid muscle damage, muscle injury or muscle atrophy is selected from thegroup consisting of acute muscle injury, tear, or trauma, soft tissuehand injury, Duchenne muscular dystrophy (DMD), Becker musculardystrophy, limb girdle muscular dystrophy, amyotrophic lateral sclerosis(ALS), distal muscular dystrophy (DD), inherited myopathies, myotonicmuscular dystrophy (MDD), mitochondrial myopathies, myotubular myopathy(MM), myasthenia gravis (MG), congestive heart failure, periodicparalysis, polymyositis, rhabdomyolysis, dermatomyositis, cancercachexia, AIDS cachexia, cardiac cachexia, stress induced urinaryincontinence, sarcopenia, and spinal muscular atrophy.
 34. The method ofclaim 31, wherein said muscle damage, muscle injury or muscle atrophy issarcopenia.
 35. The method of claim 31, wherein said muscle damage,muscle injury or muscle atrophy is Duchenne muscular dystrophy (DMD).36. The method of claim 31, wherein said muscle damage, muscle injury ormuscle atrophy is spinal muscular atrophy.
 37. The method of claim 31,wherein said administering comprises parenteral administration,intravenous administration, subcutaneous administration, intraperitonealadministration, intramuscular administration, intra-arterialadministration, intravascular administration, intracardiacadministration, intrathecal administration, intranasal administration,intradermal administration, intravitreal administration, transmucosaladministration, oral administration, administration as a suppository,and/or topical administration.
 38. The method of claim 31, wherein saidadministering comprises oral administration.
 39. The method of claim 31,wherein said composition is administered in accordance with an acuteregimen.
 40. The method of claim 31, wherein said composition isadministered in accordance with an intermittent regimen.
 41. The methodof claim 31, wherein said composition is administered in accordance witha chronic regimen.
 42. The method of claim 31, wherein said compositionfurther comprises a pharmaceutically acceptable carrier.